Isolated human transporter cofactor proteins, nucleic acid molecules encoding human transporter cofactor proteins, and uses thereof

ABSTRACT

The present invention provides amino acid sequences of peptides that are encoded by genes within the human genome, the transporter cofactor peptides of the present invention. The present invention specifically provides isolated peptide and nucleic acid molecules, methods of identifying orthologs and paralogs of the transporter cofactor peptides, and methods of identifying modulators of the transporter cofactor peptides.

FIELD OF THE INVENTION

The present invention is in the field of transporter cofactor proteins that are related to the sodium/hydrogen exchanger-regulatory factor (NHE-RF) subfamily, recombinant DNA molecules, and protein production. The present invention specifically provides novel peptides and proteins that effect ligand transport and nucleic acid molecules encoding such peptide and protein molecules, all of which are useful in the development of human therapeutics and diagnostic compositions and methods.

BACKGROUND OF THE INVENTION Transporter Regulatory Factors/Cofactors

Transporter proteins regulate many different functions of a cell, including cell proliferation, differentiation, and signaling processes, by regulating the flow of molecules such as ions and macromolecules, into and out of cells. Transporters are found in the plasma membranes of virtually every cell in eukaryotic organisms. Transporters mediate a variety of cellular functions including regulation of membrane potentials and absorption and secretion of molecules and ion across cell membranes. When present in intracellular membranes of the Golgi apparatus and endocytic vesicles, transporters, such as chloride channels, also regulate organelle pH. For a review, see Greger, R. (1988) Annu. Rev. Physiol. 50:111-122.

The novel human protein provided by the present invention is related to transporter regulatory factors, also referred to as transporter cofactors. In particular, the protein of the present invention is similar to regulatory factors of sodium/hydrogen exchangers (solute carrier family 9), referred to as sodium/hydrogen exchanger-regulatory factors (NHE-RFs). NHE-RFs participate in the protein kinase A regulation of sodium/hydrogen exchangers, such as those found at the renal brush border, and are typically found in actin-rich structures (Reczek et al., J Cell Biol Oct. 6, 1997; 139(1):169-79; Weinman et al., J Clin Invest 1995 May;95(5):2143-9). Furthermore, the protein of the present invention shows a high degree of similarity to ezrin-radixin-moesin (ERM) proteins, which are related to the NHE-RF family, particularly ERM-binding phosphoprotein 50 protein (EBP50). The ERM family of proteins are membrane-cytoskeletal linking proteins that have NH2- and COOH-terminal domains that bind the plasma membrane and the actin cytoskeleton, respectively (Reczek et al, J Cell Biol Oct. 6, 1997; 139(1):169-79).

ERM proteins are found in high concentrations in the apical portion of polarized microvilli-containing epithelial cells, which are abundant in such locations as the placenta and intestinal brush border; the actin filaments within microvilli must attach to the epithelial cell membranes in order to properly assemble and maintain the microvilli. ERM proteins are involved in linking integral membrane proteins and cytoskeletal proteins such as actin. Actin cytoskeletal assembly requires activation of sodium/hydrogen exchangers, which is modulated by NHE-RF proteins.

EBP50 binding interactions are involved in such important biological processes as membrane protein trafficking, regulating the activity of interacting proteins, and moving such interactors into common microdomains to facilitate interaction .(Fouassier et al., J Biol Chem Aug. 11, 2000; 275(32):25039-45). Additionally, a human homologue of NHE-RF has been identified as an interactor of merlin, which is a neurofibromatosis 2 tumor suppressor protein; the human NHE-RF may play a critical role in the tumor suppressor functions of merlin (Murthy et al., J Biol Chem Jan. 16, 1998; 273(3):1273-6).

EBP50 has two PSD-95/DlgA/ZO-1-like (PDZ) domains, which are known to bind integral membrane proteins (Reczek et al., J Cell Biol Oct. 6, 1997; 139(1):169-79). NHE-RF modulates protein kinase A regulation of sodium-hydrogen exchanger through binding of the PDZ domains. NHE-RF may also bind to the ERM proteins, ezrin, moesin and radixin, via the PDZ domains (Murthy et al., J Biol Chem Jan. 16, 1998; 273(3):1273-6). Furthermore, EBP50 proteins self-associate with other EBP50 proteins via their PDZ domains, providing enhanced functional capabilities, such as the ability to form multiprotein complexes and regulate membrane transport processes (Fouassier et al., J Biol Chem Aug. 11, 2000; 275(32):25039-45).

Transporter cofactors, particularly members of the sodium/hydrogen exchanger-regulatory factor subfamily, are a major target for drug action and development. Accordingly, it is valuable to the field of pharmaceutical development to identify and characterize previously unknown transport proteins. The present invention advances the state of the art by providing previously unidentified human transport proteins.

SUMMARY OF THE INVENTION

The present invention is based in part on the identification of amino acid sequences of human transporter cofactor peptides and proteins that are related to the sodium/hydrogen exchanger-regulatory factor subfamily, as well as allelic variants and other mammalian orthologs thereof. These unique peptide sequences, and nucleic acid sequences that encode these peptides, can be used as models for the development of human therapeutic targets, aid in the identification of therapeutic proteins, and serve as targets for the development of human therapeutic agents that modulate transporter cofactor activity in cells and tissues that express the transporter cofactor. Experimental data as provided in FIG. 1 indicates expression in humans in placenta choriocarcinomas, ovary adenocarcinomas, retinoblastomas of the eye, brain neuroblastomas, endometrium adenocarcinomas, colon, lung small cell carcinomas, T-lymphocytes, ovarian tumors, pheochromocytomas, fetal liver/spleen, Burkitt's lymphoma, and leukocytes.

DESCRIPTION OF THE FIGURE SHEETS

FIG. 1 (page 1 of 2, page 2 of 2) provides the nucleotide sequence of a cDNA molecule that encodes the transporter cofactor protein of the present invention. In addition structure and functional information is provided, such as ATG start, stop and tissue distribution, where available, that allows one to readily determine specific uses of inventions based on this molecular sequence. Experimental data as provided in FIG. 1 indicates expression in humans in placenta choriocarcinomas, ovary adenocarcinomas, retinoblastomas of the eye, brain neuroblastomas, endometrium adenocarcinomas, colon, lung small cell carcinomas, T-lymphocytes, ovarian tumors, pheochromocytomas, fetal liver/spleen, Burkitt's lymphoma, and leukocytes.

FIG. 2 (page 1 of 2, page 2 of 2) provides the predicted amino acid sequence of the transporter cofactor of the present invention. In addition structure and functional information such as protein family, function, and modification sites is provided where available, allowing one to readily determine specific uses of inventions based on this molecular sequence.

FIG. 3 (page 1 of 12 through page 12 of 12) provides genomic sequences that span the gene encoding the transporter cofactor protein of the present invention. In addition structure and functional information, such as intron/exon structure, promoter location, etc., is provided where available, allowing one to readily determine specific uses of inventions based on this molecular sequence. As illustrated in FIG. 3 (page 1 of 12 through page 12 of 12), SNPs were identified at 18 different nucleotide positions.

DETAILED DESCRIPTION OF THE INVENTION General Description

The present invention is based on the sequencing of the human genome. During the sequencing and assembly of the human genome, analysis of the sequence information revealed previously unidentified fragments of the human genome that encode peptides that share structural and/or sequence homology to protein/peptide/domains identified and characterized within the art as being a transporter cofactor protein or part of a transporter cofactor protein and are related to the sodium/hydrogen exchanger-regulatory factor subfamily. Utilizing these sequences, additional genomic sequences were assembled and transcript and/or cDNA sequences were isolated and characterized. Based on this analysis, the present invention provides amino acid sequences-of human transporter cofactor peptides and proteins that are related to the sodium/hydrogen exchanger-regulatory factor subfamily, nucleic acid sequences in the form of transcript sequences, cDNA sequences and/or genomic sequences that encode these transporter cofactor peptides and proteins, nucleic acid variation (allelic information), tissue distribution of expression, and information about the closest art known protein/peptide/domain that has structural or sequence homology to the transporter cofactor of the present invention.

In addition to being previously unknown, the peptides that are provided in the present invention are selected based on their ability to be used for the development of commercially important products and services. Specifically, the present peptides are selected based on homology and/or structural relatedness to known transporter cofactor proteins of the sodium/hydrogen exchanger-regulatory factor subfamily and the expression pattern observed. Experimental data as provided in FIG. 1 indicates expression in humans in placenta choriocarcinomas, ovary adenocarcinomas, retinoblastomas of the eye, brain neuroblastomas, endometrium adenocarcinomas, colon, lung small cell carcinomas, T-lymphocytes, ovarian tumors, pheochromocytomas, fetal liver/spleen, Burkitt's lymphoma, and leukocytes. The art has clearly established the commercial importance of members of this family of proteins and proteins that have expression patterns similar to that of the present gene. Some of the more specific features of the peptides of the present invention, and the uses thereof, are described herein, particularly in the Background of the Invention and in the annotation provided in the Figures, and/or are known within the art for each of the known sodium/hydrogen exchanger-regulatory factor family or subfamily of transporter cofactor proteins.

Specific Embodiments

Peptide Molecules

The present invention provides nucleic acid sequences that encode protein molecules that have been identified as being members of the transporter cofactor family of proteins and are related to the sodium/hydrogen exchanger-regulatory factor subfamily (protein sequences are provided in FIG. 2, transcript/cDNA sequences are provided in FIG. 1 and genomic sequences are provided in FIG. 3). The peptide sequences provided in FIG. 2, as well as the obvious variants described herein, particularly allelic variants as identified herein and using the information in FIG. 3, will be referred herein as the transporter cofactor peptides of the present invention, transporter cofactor peptides, or peptides/proteins of the present invention.

The present invention provides isolated peptide and protein molecules that consist of, consist essentially of, or comprising the amino acid sequences of the transporter cofactor peptides disclosed in the FIG. 2, (encoded by the nucleic acid molecule shown in FIG. 1, transcript/cDNA or FIG. 3, genomic sequence), as well as all obvious variants of these peptides that are within the art to make and use. Some of these variants are described in detail below.

As used herein, a peptide is said to be “isolated” or “purified” when it is substantially free of cellular material or free of chemical precursors or other chemicals. The peptides of the present invention can be purified to homogeneity or other degrees of purity. The level of purification will be based on the intended use. The critical feature is that the preparation allows for the desired function of the peptide, even if in the presence of considerable amounts of other components (the features of an isolated nucleic acid molecule is discussed below).

In some uses, “substantially free of cellular material” includes preparations of the peptide having less than about 30% (by dry weight) other proteins (i.e., contaminating protein), less than about 20% other proteins, less than about 10% other proteins, or less than about 5% other proteins. When the peptide is recombinantly produced, it can also be substantially free of culture medium, i.e., culture medium represents less than about 20% of the volume of the protein preparation.

The language “substantially free of chemical precursors or other chemicals” includes preparations of the peptide in which it is separated from chemical precursors or other chemicals that are involved in its synthesis. In one embodiment, the language “substantially free of chemical precursors or other chemicals” includes preparations of the transporter cofactor peptide having less than about 30% (by dry weight) chemical precursors or other chemicals, less than about 20% chemical precursors or other chemicals, less than about 10% chemical precursors or other chemicals, or less than about 5% chemical precursors or other chemicals.

The isolated transporter cofactor peptide can be purified from cells that naturally express it, purified from cells that have been altered to express it (recombinant), or synthesized using known protein synthesis methods. Experimental data as provided in FIG. 1 indicates expression in humans in placenta choriocarcinomas, ovary adenocarcinomas, retinoblastomas of the eye, brain neuroblastomas, endometrium adenocarcinomas, colon, lung small cell carcinomas, T-lymphocytes, ovarian tumors, pheochromocytomas, fetal liver/spleen, Burkitt's lymphoma, and leukocytes. For example, a nucleic acid molecule encoding the transporter cofactor peptide is cloned into an expression vector, the expression vector introduced into a host cell and the protein expressed in the host cell. The protein can then be isolated from the cells by an appropriate purification scheme using standard protein purification techniques. Many of these techniques are described in detail below.

Accordingly, the present invention provides proteins that consist of the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example, proteins encoded by the transcript/cDNA nucleic acid sequences shown in FIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG. 3 (SEQ ID NO:3). The amino acid sequence of such a protein is provided in FIG. 2. A protein consists of an amino acid sequence when the amino acid sequence is the final amino acid sequence of the protein.

The present invention further provides proteins that consist essentially of the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example, proteins encoded by the transcript/cDNA nucleic acid sequences shown in FIG. 1 (SEQ ID NO: 1) and the genomic sequences provided in FIG. 3 (SEQ ID NO:3). A protein consists essentially of an amino acid sequence when such an amino acid sequence is present with only a few additional amino acid residues, for example from about 1 to about 100 or so additional residues, typically from 1 to about 20 additional residues in the final protein.

The present invention further provides proteins that comprise the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example, proteins encoded by the transcript/cDNA nucleic acid sequences shown in FIG. 1 (SEQ ID NO: 1) and the genomic sequences provided in FIG. 3 (SEQ ID NO:3). A protein comprises an amino acid sequence when the amino acid sequence is at least part of the final amino acid sequence of the protein. In such a fashion, the protein can be only the peptide or have additional amino acid molecules, such as amino acid residues (contiguous encoded sequence) that are naturally associated with it or heterologous amino acid residues/peptide sequences. Such a protein can have a few additional amino acid residues or can comprise several hundred or more additional amino acids. The preferred classes of proteins that are comprised of the transporter cofactor peptides of the present invention are the naturally occurring mature proteins. A brief description of how various types of these proteins can be made/isolated is provided below.

The transporter cofactor peptides of the present invention can be attached to heterologous sequences to form chimeric or fusion proteins. Such chimeric and fusion proteins comprise a transporter cofactor peptide operatively linked to a heterologous protein having an amino acid sequence not substantially homologous to the transporter cofactor peptide. “Operatively linked” indicates that the transporter cofactor peptide and the heterologous protein are fused in-frame. The heterologous protein can be fused to the N-terminus or C-terminus of the transporter cofactor peptide.

In some uses, the fusion protein does not affect the activity of the transporter cofactor peptide per se. For example, the fusion protein can include, but is not limited to, enzymatic fusion proteins, for example beta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-His fusions, MYC-tagged, HI-tagged and Ig fusions. Such fusion proteins, particularly poly-His fusions, can facilitate the purification of recombinant transporter cofactor peptide. In certain host cells (e.g., mammalian host cells), expression and/or secretion of a protein can be increased by using a heterologous signal sequence.

A chimeric or fusion protein can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different protein sequences are ligated together in-frame in accordance with conventional techniques. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and re-amplified to generate a chimeric gene sequence (see Ausubel et al., Current Protocols in Molecular Biology, 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST protein). A transporter cofactor peptide-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the transporter cofactor peptide.

As mentioned above, the present invention also provides and enables obvious variants of the amino acid sequence of the proteins of the present invention, such as naturally occurring mature forms of the peptide, allelic/sequence variants of the peptides, non-naturally occurring recombinantly derived variants of the peptides, and orthologs and paralogs of the peptides. Such variants can readily be generated using art-known techniques in the fields of recombinant nucleic acid technology and protein biochemistry. It is understood, however, that variants exclude any amino acid sequences disclosed prior to the invention.

Such variants can readily be identified/made using molecular techniques and the sequence information disclosed herein. Further, such variants can readily be distinguished from other peptides based on sequence and/or structural homology to the transporter cofactor peptides of the present invention. The degree of homology/identity present will be based primarily on whether the peptide is a functional variant or non-functional variant, the amount of divergence present in the paralog family and the evolutionary distance between, the orthologs.

To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more of a reference sequence is aligned for comparison purposes. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

The comparison of sequences and determination of percent identity and similarity between two sequences can be accomplished using a mathematical algorithm. (Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (Devereux, J., et al., Nucleic Acids Res. 12(1):387 (1984)) (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of E. Myers and W. Miller (CABIOS, 4:11 -17 (1989)) which has been incorporated into the ALIGN program (version 2.0), using a, PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

The nucleic acid and protein sequences of the present invention can further be used as a “query sequence” to perform a search against sequence databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (J. Mol. Biol. 215:403-10 (1990)). BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the proteins of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al (Nucleic Acids Res. 25(17):3389-3402 (1997). When utilizing BLAST and gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

Full-length pre-processed forms, as well as mature processed forms, of proteins that comprise one of the peptides of the present invention can readily be identified as having complete sequence identity to one of the transporter cofactor peptides of the present invention as well as being encoded by the same genetic locus as the transporter cofactor peptide provided herein. The gene encoding the novel transporter regulatory factor protein of the present invention is located on a genome component that has been mapped to human chromosome 17 (as indicated in FIG. 3), which is supported by multiple lines of evidence, such as STS and BAC map data.

Allelic variants of a transporter cofactor peptide can readily be identified as being a human protein having a high degree (significant) of sequence homology/identity to at least a portion of the transporter cofactor peptide as well as being encoded by the same genetic locus as the transporter cofactor peptide provided herein. Genetic locus can readily be determined based on the genomic information provided in FIG. 3, such as the genomic sequence mapped to the reference human. The gene encoding the novel transporter regulatory factor protein of the present invention is located on a genome component that has been mapped to human chromosome 17 (as indicated in FIG. 3), which is supported by multiple lines of evidence, such as STS and BAC map data. As used herein, two proteins (or a region of the proteins) have significant homology when the amino acid sequences are typically at least about 70-80%, 80-90%, and more typically at least about 90-95% or more homologous. A significantly homologous amino acid sequence, according to the present invention, will be encoded by a nucleic acid sequence that will hybridize to a transporter cofactor peptide encoding nucleic acid molecule under stringent conditions as more fully described below.

FIG. 3 provides information on SNPs that have been found in the gene encoding the transporter cofactor protein of the present invention. SNPs were identified at 18 different nucleotide positions. Changes in the amino acid sequence caused by these SNPs can readily be determined using the universal genetic code and the protein sequence provided in FIG. 2 as a reference. SNPs outside the ORF and in introns may affect control/regulatory elements. Positioning of each SNP in exons, introns, or outside the ORF can readily be determined using the DNA positions given for each SNP and the start/stop, exon, and intron genomic coordinates given in FIG. 3.

Paralogs of a transporter cofactor peptide can readily be identified as having some degree of significant sequence homology/identity to at least a portion of the transporter cofactor peptide, as being encoded by a gene from humans, and as having similar activity or function. Two proteins will typically be considered paralogs when the amino acid sequences are typically at least about 60% or greater, and more typically at least about 70% or greater homology through a given region or domain. Such paralogs will be encoded by a nucleic acid sequence that will hybridize to a transporter cofactor peptide encoding nucleic acid molecule under moderate to stringent conditions as more fully described below.

Orthologs of a transporter cofactor peptide can readily be identified as having some degree of significant sequence homology/identity to at least a portion of the transporter cofactor peptide as well as being encoded by a gene from another organism. Preferred orthologs will be isolated from mammals, preferably primates, for the development of human therapeutic targets and agents. Such orthologs will be encoded by a nucleic acid sequence that will hybridize to a transporter cofactor peptide encoding nucleic acid molecule under moderate to stringent conditions, as more fully described below, depending on the degree of relatedness of the two organisms yielding the proteins.

Non-naturally occurring variants of the transporter cofactor peptides of the present invention can readily be generated using recombinant techniques. Such variants include, but are not limited to deletions, additions and substitutions in the amino acid sequence of the transporter cofactor peptide. For example, one class of substitutions are conserved amino acid substitution. Such substitutions are those that substitute a given amino acid in a transporter cofactor peptide by another amino acid of like characteristics. Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu, and Ile; interchange of the hydroxyl residues Ser and Thr; exchange of the acidic residues Asp and Glu; substitution between the amide residues Asn and Gln; exchange of the basic residues Lys and Arg; and replacements among the aromatic residues Phe and Tyr. Guidance concerning which amino acid changes are likely to be phenotypically silent are found in Bowie et al., Science 247:1306-1310 (1990).

Variant transporter cofactor peptides can be fully functional or can lack function in one or more activities, e.g. ability to bind ligand, ability to transport ligand, ability to mediate signaling, etc. Fully functional variants typically contain only conservative variation or variation in non-critical residues or in non-critical regions. FIG. 2 provides the result of protein analysis and can be used to identify critical domains/regions. Functional variants can also contain substitution of similar amino acids that result in no change or an insignificant change in function. Alternatively, such substitutions may positively or negatively affect function to some degree.

Non-functional variants typically contain one or more non-conservative amino acid substitutions, deletions, insertions, inversions, or truncation or a substitution, insertion, inversion, or deletion in a critical residue or critical region.

Amino acids that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham et al., Science 244:1081-1085 (1989)), particularly using the results provided in FIG. 2. The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity such as transporter cofactor activity or in assays such as an in vitro proliferative activity. Sites that are critical for binding partner/substrate binding can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al., J Mol. Biol. 224:899-904 (1992); de Vos et al. Science 255:306-312 (1992)).

The present invention further provides fragments of the transporter cofactor peptides, in addition to proteins and peptides that comprise and consist of such fragments, particularly those comprising the residues identified in FIG. 2. The fragments to which the invention pertains, however, are not to be construed as encompassing fragments that may be disclosed publicly prior to the present invention.

As used herein, a fragment comprises at least 8, 10, 12, 14, 16, or more contiguous amino acid residues from a transporter cofactor peptide. Such fragments can be chosen based on the ability to retain one or more of the biological activities of the transporter cofactor peptide or could be chosen for the ability to perform a function, e.g. bind a substrate or act as an immunogen. Particularly important fragments are biologically active fragments, peptides that are, for example, about 8 or more amino acids in length. Such fragments will typically comprise a domain or motif of the transporter cofactor peptide, e.g., active site, a transmembrane domain or a substrate-binding domain. Further, possible fragments include, but are not limited to, domain or motif containing fragments, soluble peptide fragments, and fragments containing immunogenic structures. Predicted domains and functional sites are readily identifiable by computer programs well known and readily available to those of skill in the art (e.g., PROSITE analysis). The results of one such analysis are provided in FIG. 2.

Polypeptides often contain amino acids other than the 20 amino acids commonly referred to as the 20 naturally occurring amino acids. Further, many amino acids, including the terminal amino acids, may be modified by natural processes, such as processing and other post-translational modifications, or by chemical modification techniques well known in the art. Common modifications that occur naturally in transporter cofactor peptides are described in basic texts, detailed monographs, and the research literature, and they are well known to those of skill in the art (some of these features are identified in FIG. 2).

Known modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.

Such modifications are well known to those of skill in the art and have been described in great detail in the scientific literature. Several particularly common modifications, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation, for instance, are described in most basic texts, such as Proteins—Structure and Molecular Properties, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993). Many detailed reviews are available on this subject, such as by Wold, F., Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York 1-12 (1983); Seifter et al. (Meth. Enzymol. 182:626-646 (1990)) and Rattan et al. (Ann. N.Y. Acad. Sci. 663:48-62 (1992)).

Accordingly, the transporter cofactor peptides of the present invention also encompass derivatives or analogs in which a substituted amino acid residue is not one encoded by the genetic code, in which a substituent group is included, in which the mature transporter cofactor peptide is fused with another compound, such as a compound to increase the half-life of the transporter cofactor peptide (for example, polyethylene glycol), or in which the additional amino acids are fused to the mature transporter cofactor peptide, such as a leader or secretory sequence or a sequence for purification of the mature transporter cofactor peptide or a pro-protein sequence.

Protein/Peptide Uses

The proteins of the present invention can be used in substantial and specific assays related to the functional information provided in the Figures; to raise antibodies or to elicit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its binding partner or ligand) in biological fluids; and as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state). Where the protein binds or potentially binds to another protein or ligand (such as, for example, in a transporter cofactor-effector protein interaction or transporter cofactor-ligand interaction), the protein can be used to identify the binding partner/ligand so as to develop a system to identify inhibitors of the binding interaction. Any or all of these uses are capable of being developed into reagent grade or kit format for commercialization as commercial products.

Methods for performing the uses listed above are well known to those skilled in the art. References disclosing such methods include “Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds., 1989, and “Methods in Enzymology: Guide to Molecular Cloning Techniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

The potential uses of the peptides of the present invention are based primarily on the source of the protein as well as the class/action of the protein. For example, transporter cofactors isolated from humans and their human/mammalian orthologs serve as targets for identifying agents for use in mammalian therapeutic applications, e.g. a human drug, particularly in modulating a biological or pathological response in a cell or tissue that expresses the transporter cofactor. Experimental data as provided in FIG. 1 indicates that the transporter cofactor proteins of the present invention are expressed in humans in placenta choriocarcinomas, ovary adenocarcinomas, retinoblastomas of the eye, brain neuroblastomas, endometrium adenocarcinomas, colon, lung small cell carcinomas, T-lymphocytes, ovarian tumors, pheochromocytomas, fetal liver/spleen, and Burkitt's lymphoma, as indicated by virtual northern blot analysis. In addition, PCR-based tissue screening panels indicate expression in human leukocytes. A large percentage of pharmaceutical agents are being developed that modulate the activity of transporter cofactor proteins, particularly members of the sodium/hydrogen exchanger-regulatory factor subfamily (see Background of the Invention). The structural and functional information provided in the Background and Figures provide specific and substantial uses for the molecules of the present invention, particularly in combination with the expression information provided in FIG. 1. Experimental data as provided in FIG. 1 indicates expression in humans in placenta choriocarcinomas, ovary adenocarcinomas, retinoblastomas of the eye, brain neuroblastomas, endometrium adenocarcinomas, colon, lung small cell carcinomas, T-lymphocytes, ovarian tumors, pheochromocytomas, fetal liver/spleen, Burkitt's lymphoma, and leukocytes. Such uses can readily be determined using the information provided herein, that known in the art and routine experimentation.

The proteins of the present invention (including variants and fragments that may have been disclosed prior to the present invention) are useful for biological assays related to transporter cofactors that are related to members of the sodium/hydrogen exchanger-regulatory factor subfamily. Such assays involve any of the known transporter cofactor functions or activities or properties useful for diagnosis and treatment of transporter cofactor-related conditions that are specific for the subfamily of transporter cofactors that the one of the present invention belongs to, particularly in cells and tissues that express the transporter cofactor. Experimental data as provided in FIG. 1 indicates that the transporter cofactor proteins of the present invention are expressed in humans in placenta choriocarcinomas, ovary adenocarcinomas, retinoblastomas of the eye, brain neuroblastomas, endometrium adenocarcinomas, colon, lung small cell carcinomas, T-lymphocytes, ovarian tumors, pheochromocytomas, fetal liver/spleen, and Burkitt's lymphoma, as indicated by virtual northern blot analysis. In addition, PCR-based tissue screening panels indicate expression in human leukocytes. The proteins of the present invention are also useful in drug screening assays, in cell-based or cell-free systems (Hodgson, Bio/technology, 1992, Sep. 10(9);973-80). Cell-based systems can be native, i.e., cells that normally express the transporter cofactor, as a biopsy or expanded in cell culture. Experimental data as provided in FIG. 1 indicates expression in humans in a placenta choriocarcinomas, ovary adenocarcinomas, retinoblastomas of the eye, brain neuroblastomas, endometrium adenocarcinomas, colon, lung small cell carcinomas, T-lymphocytes, ovarian tumors, pheochromocytomas, fetal liver/spleen, Burkitt's lymphoma, and leukocytes. In an alternate embodiment, cell-based assays involve recombinant host cells expressing the transporter cofactor protein.

The polypeptides can be used to identify compounds that modulate transporter cofactor activity of the protein in its natural state or an altered form that causes a specific disease or pathology associated with the transporter cofactor. Both the transporter cofactors of the present invention and appropriate variants and fragments can be used in high-thoughput screens to assay candidate compounds for the ability to bind to the transporter cofactor. These compounds can be further screened against a functional transporter cofactor to determine the effect of the compound on the transporter cofactor activity. Further, these compounds can be tested in animal or invertebrate systems to determine activity/effectiveness. Compounds can be identified that activate (agonist) or inactivate (antagonist) the transporter cofactor to a desired degree.

Further, the proteins of the present invention can be used to screen a compound for the ability to stimulate or inhibit interaction between the transporter cofactor protein and a molecule that normally interacts with the transporter cofactor protein, e.g. a substrate or a component of the signal pathway that the transporter cofactor protein normally interacts (for example, a sodum/hydrogen exchanger). Such assays typically include the steps of combining the transporter cofactor protein with a candidate compound under conditions that allow the transporter cofactor protein, or fragment, to interact with the target molecule, and to detect the formation of a complex between the protein and the target or to detect the biochemical consequence of the interaction with the transporter cofactor protein and the target, such as any of the associated effects of signal transduction such as changes in membrane potential, protein phosphorylation, cAMP turnover, and adenylate cyclase activation, etc.

Candidate compounds include, for example, 1) peptides such as soluble peptides, including Ig-tailed fusion peptides and members of random peptide libraries (see, e.g., Lam et al., Nature 354:82-84 (1991); Houghten et al., Nature 354:84-86 (1991)) and combinatorial chemistry-derived molecular libraries made of D- and/or L-configuration amino acids; 2) phosphopeptides (e.g., members of random and partially degenerate, directed phosphopeptide libraries, see, e.g., Songyang et al., Cell 72:767-778 (1993)); 3) antibodies (e.g., polyclonal, monoclonal, humanized, anti-idiotypic, chimeric, and single chain antibodies as well as Fab, F(ab′)₂, Fab expression library fragments, and epitope-binding fragments of antibodies); and 4) small organic and inorganic molecules (e.g., molecules obtained from combinatorial and natural product libraries).

One candidate compound is a soluble fragment of the receptor that competes for ligand binding. Other candidate compounds include mutant transporter cofactors or appropriate fragments containing mutations that affect transporter cofactor function and thus compete for ligand. Accordingly, a fragment that competes for ligand, for example with a higher affinity, or a fragment that binds ligand but does not allow release, is encompassed by the invention.

The invention further includes other end point assays to identify compounds that modulate (stimulate or inhibit) transporter cofactor activity. The assays typically involve an assay of events in the signal transduction pathway that indicate transporter cofactor activity. Thus, the transport of a ligand, change in cell membrane potential, activation of a protein, a change in the expression of genes that are up- or down-regulated in response to the transporter cofactor protein dependent signal cascade can be assayed.

Any of the biological or biochemical functions mediated by the transporter cofactor can be used as an endpoint assay. These include all of the biochemical or biochemical/biological events described herein, in the references cited herein, incorporated by reference for these endpoint assay targets, and other functions known to those of ordinary skill in the art or that can be readily identified using the information provided in the Figures, particularly FIG. 2. Specifically, a biological function of a cell or tissues that expresses the transporter cofactor can be assayed. Experimental data as provided in FIG. 1 indicates that the transporter cofactor proteins of the present invention are expressed in humans in placenta choriocarcinomas, ovary adenocarcinomas, retinoblastomas of the eye, brain neuroblastomas, endometrium adenocarcinomas, colon, lung small cell carcinomas, T-lymphocytes, ovarian tumors, pheochromocytomas, fetal liver/spleen, and Burkitt's lymphoma, as indicated by virtual northern blot analysis. In addition, PCR-based tissue screening panels indicate expression in human leukocytes.

Binding and/or activating compounds can also be screened by using chimeric transporter cofactor proteins in which the amino terminal extracellular domain, or parts thereof, the entire transmembrane domain or subregions, such as any of the seven transmembrane segments or any of the intracellular or extracellular loops and the carboxy terminal intracellular domain, or parts thereof, can be replaced by heterologous domains or subregions. For example, a ligand-binding region can be used that interacts with a different ligand then that which is recognized by the native transporter cofactor. Accordingly, a different set of signal transduction components is available as an end-point assay for activation. This allows for assays to be performed in other than the specific host cell from which the transporter cofactor is derived.

The proteins of the present invention are also useful in competition binding assays in methods designed to discover compounds that interact with the transporter cofactor (e.g. binding partners and/or ligands). Thus, a compound is exposed to a transporter cofactor polypeptide under conditions that allow the compound to bind or to otherwise interact with the polypeptide. Soluble transporter cofactor polypeptide is also added to the mixture. If the test compound interacts with the soluble transporter cofactor polypeptide, it decreases the amount of complex formed or activity from the transporter cofactor target. This type of assay is particularly useful in cases in which compounds are sought that interact with specific regions of the transporter cofactor. Thus, the soluble polypeptide that competes with the target transporter cofactor region is designed to contain peptide sequences corresponding to the region of interest.

To perform cell free drug screening assays, it is sometimes desirable to immobilize either the transporter cofactor protein, or fragment, or its target molecule to facilitate separation of complexes from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay.

Techniques for immobilizing proteins on matrices can be used in the drug screening assays. In one embodiment, a fusion protein can be provided which adds a domain that allows the protein to be bound to a matrix. For example, glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtitre plates, which are then combined with the cell lysates (e.g., ³⁵S-labeled) and the candidate compound, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads are washed to remove any unbound label, and the matrix immobilized and radiolabel determined directly, or in the supernatant after the complexes are dissociated. Alternatively, the complexes can be dissociated from the matrix, separated by SDS-PAGE, and the level of transporter cofactor-binding protein found in the bead fraction quantitated from the gel using standard electrophoretic techniques. For example, either the polypeptide or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin using techniques well known in the art. Alternatively, antibodies reactive with the protein but which do not interfere with binding of the protein to its target molecule can be derivatized to the wells of the plate, and the protein trapped in the wells by antibody conjugation. Preparations of a transporter cofactor-binding protein and a candidate compound are incubated in the transporter cofactor protein-presenting wells and the amount of complex trapped in the well can be quantitated. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the transporter cofactor protein target molecule, or which are reactive with transporter cofactor protein and compete with the target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the target molecule.

Agents that modulate one of the transporter cofactors of the present invention can be identified using one or more of the above assays, alone or in combination. It is generally preferable to use a cell-based or cell free system first and then confirm activity in an animal or other model system. Such model systems are well known in the art and can readily be employed in this context.

Modulators of transporter cofactor activity identified according to these drug screening assays can be used to treat a subject with a disorder mediated by the transporter cofactor pathway, by treating cells or tissues that express the transporter cofactor. Experimental data as provided in FIG. 1 indicates expression in humans in placenta choriocarcinomas, ovary adenocarcinomas, retinoblastomas of the eye, brain neuroblastomas, endometrium adenocarcinomas, colon, lung small cell carcinomas, T-lymphocytes, ovarian tumors, pheochromocytomas, fetal liver/spleen, Burkitt's lymphoma, and leukocytes. These methods of treatment include the steps of administering a modulator of transporter cofactor activity in a pharmaceutical composition to a subject in need of such treatment, the modulator being identified as described herein.

In yet another aspect of the invention, the transporter cofactors can be used as “bait proteins” in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent WO94/10300), to identify other proteins, which bind to or interact with the transporter cofactor and are involved in transporter cofactor activity. Such transporter cofactor-binding proteins are also likely to be involved in the propagation of signals by the transporter cofactors or transporter cofactor targets as, for example, downstream elements of a transporter cofactor-mediated signaling pathway. Alternatively, such transporter cofactor-binding proteins are likely to be transporter cofactor inhibitors.

The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for a transporter cofactor protein is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein (“prey” or “sample”) is fused to a gene that codes for the activation domain of the known transcription factor. If the “bait” and the “prey” proteins are able to interact, in vivo, forming a transporter cofactor-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with the transporter cofactor protein.

This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in an appropriate animal model. For example, an agent identified as described herein (e.g., a transporter cofactor-modulating agent, an antisense transporter cofactor nucleic acid molecule, a transporter cofactor-specific antibody, or a transporter cofactor-binding partner) can be used in an animal or other model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an agent identified as described herein can be used in an animal or other model to determine the mechanism of action of such an agent. Furthermore, this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein.

The transporter cofactor proteins of the present invention are also useful to provide a target for diagnosing a disease or predisposition to disease mediated by the peptide. Accordingly, the invention provides methods for detecting the presence, or levels of, the protein (or encoding mRNA) in a cell, tissue, or organism. Experimental data as provided in FIG. 1 indicates expression in humans in placenta choriocarcinomas, ovary adenocarcinomas, retinoblastomas of the eye, brain neuroblastomas, endometrium adenocarcinomas, colon, lung small cell carcinomas, T-lymphocytes, ovarian tumors, pheochromocytomas, fetal liver/spleen, Burkitt's lymphoma, and leukocytes. The method involves contacting a biological sample with a compound capable of interacting with the transporter cofactor protein such that the interaction can be detected. Such an assay can be provided in a single detection format or a multi-detection format such as an antibody chip array.

One agent for detecting a protein in a sample is an antibody capable of selectively binding to protein. A biological sample includes tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject.

The peptides of the present invention also provide targets for diagnosing active protein activity, disease, or predisposition to disease, in a patient having a variant peptide, particularly activities and conditions that are known for other members of the family of proteins to which the present one belongs. Thus, the peptide can be isolated from a biological sample and assayed for the presence of a genetic mutation that results in aberrant peptide. This includes amino acid substitution, deletion, insertion, rearrangement, (as the result of aberrant splicing events), and inappropriate post-translational modification. Analytic methods include altered electrophoretic mobility, altered tryptic peptide digest, altered transporter cofactor activity in cell-based or cell-free assay, alteration in ligand or antibody-binding pattern, altered isoelectric point, direct amino acid sequencing, and any other of the known assay techniques useful for detecting mutations in a protein. Such an assay can be provided in a single detection format or a multi-detection format such as an antibody chip array.

In vitro techniques for detection of peptide include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence using a detection reagent, such as an antibody or protein binding agent. Alternatively, the peptide can be detected in vivo in a subject by introducing into the subject a labeled anti-peptide antibody or other types of detection agent. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. Particularly useful are methods that detect the allelic variant of a peptide expressed in a subject and methods which detect fragments of a peptide in a sample.

The peptides are also useful in pharmacogenomic analysis. Pharmacogenomics deal with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See, e.g., Eichelbaum, M. (Clin. Exp. Pharmacol. Physiol. 23(10-11):983-985 (1996)), and Linder, M. W. (Clin. Chem. 43(2):254-266 (1997)). The clinical outcomes of these variations result in severe toxicity of therapeutic drugs in certain individuals or therapeutic failure of drugs in certain individuals as a result of individual variation in metabolism. Thus, the genotype of the individual can determine the way a therapeutic compound acts on the body or the way the body metabolizes the compound. Further, the activity of drug metabolizing enzymes effects both the intensity and duration of drug action. Thus, the pharmacogenomics of the individual permit the selection of effective compounds and effective dosages of such compounds for prophylactic or therapeutic treatment based on the individual's genotype. The discovery of genetic polymorphisms in some drug metabolizing enzymes has explained why some patients do not obtain the expected drug effects, show an exaggerated drug effect, or experience serious toxicity from standard drug dosages. Polymorphisms can be expressed in the phenotype of the extensive metabolizer and the phenotype of the poor metabolizer. Accordingly, genetic polymorphism may lead to allelic protein variants of the transporter cofactor protein in which one or more of the transporter cofactor functions in one population is different from those in another population. The peptides thus allow a target to ascertain a genetic predisposition that can affect treatment modality. Thus, in a ligand-based treatment, polymorphism may give rise to amino terminal extracellular domains and/or other ligand-binding regions that are more or less active in ligand binding, and transporter cofactor activation. Accordingly, ligand dosage would necessarily be modified to maximize the therapeutic effect within a given population containing a polymorphism. As an alternative to genotyping, specific polymorphic peptides could be identified.

The peptides are also useful for treating a disorder characterized by an absence of, inappropriate, or unwanted expression of the protein. Experimental data as provided in FIG. 1 indicates expression in humans in placenta choriocarcinomas, ovary adenocarcinomas, retinoblastomas of the eye, brain neuroblastomas, endometrium adenocarcinomas, colon, lung small cell carcinomas, T-lymphocytes, ovarian tumors, pheochromocytomas, fetal liver/spleen, Burkitt's lymphoma, and leukocytes. Accordingly, methods for treatment include the use of the transporter cofactor protein or fragments.

Antibodies

The invention also provides antibodies that selectively bind to one of the peptides of the present invention, a protein comprising such a peptide, as well as variants and fragments thereof. As used herein, an antibody selectively binds a target peptide when it binds the target peptide and does not significantly bind to unrelated proteins. An antibody is still considered to selectively bind a peptide even if it also binds to other proteins that are not substantially homologous with the target peptide so long as such proteins share homology with a fragment or domain of the peptide target of the antibody. In this case, it would be understood that antibody binding to the peptide is still selective despite some degree of cross-reactivity.

As used herein, an antibody is defined in terms consistent with that recognized within the art: they are multi-subunit proteins produced by a mammalian organism in response to an antigen challenge. The antibodies of the present invention include polyclonal antibodies and monoclonal antibodies, as well as fragments of such antibodies, including, but not limited to, Fab or F(ab′)₂, and Fv fragments.

Many methods are known for generating and/or identifying antibodies to a given target peptide. Several such methods are described by Harlow, Antibodies, Cold Spring Harbor Press, (1989).

In general, to generate antibodies, an isolated peptide is used as an immunogen and is administered to a mammalian organism, such as a rat, rabbit or mouse. The full-length protein, an antigenic peptide fragment or a fusion protein can be used. Particularly important fragments are those covering functional domains, such as the domains identified in FIG. 2, and domain of sequence homology or divergence amongst the family, such as those that can readily be identified using protein alignment methods and as presented in the Figures.

Antibodies are preferably prepared from regions or discrete fragments of the transporter cofactor proteins. Antibodies can be prepared from any region of the peptide as described herein. However, preferred regions will include those involved in function/activity and/or transporter cofactor/binding partner interaction. FIG. 2 can be used to identify particularly important regions while sequence alignment can be used to identify conserved and unique sequence fragments.

An antigenic fragment will typically comprise at least 8 contiguous amino acid residues. The antigenic peptide can comprise, however, at least 10, 12, 14, 16 or more amino acid residues. Such fragments can be selected on a physical property, such as fragments correspond to regions that are located on the surface of the protein, e.g., hydrophilic regions or can be selected based on sequence uniqueness (see FIG. 2).

Detection on an antibody of the present invention can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or ³H.

Antibody Uses

The antibodies can be used to isolate one of the proteins of the present invention by standard techniques, such as affinity chromatography or immunoprecipitation. The antibodies can facilitate the purification of the natural protein from cells and recombinantly produced protein expressed in host cells. In addition, such antibodies are useful to detect the presence of one of the proteins of the present invention in cells or tissues to determine the pattern of expression of the protein among various tissues in an organism and over the course of normal development. Experimental data as provided in FIG. 1 indicates that the transporter cofactor proteins of the present invention are expressed in humans in placenta choriocarcinomas, ovary adenocarcinomas, retinoblastomas of the eye, brain neuroblastomas, endometrium adenocarcinomas, colon, lung small cell carcinomas, T-lymphocytes, ovarian tumors, pheochromocytomas, fetal liver/spleen, and Burkitt's lymphoma, as indicated by virtual northern blot analysis. In addition, PCR-based tissue screening panels indicate expression in human leukocytes. Further, such antibodies can be used to detect protein in situ, in vitro, or in a cell lysate or supernatant in order to evaluate the abundance and pattern of expression. Also, such antibodies can be used to assess abnormal tissue distribution or abnormal expression during development or progression of a biological condition. Antibody detection of circulating fragments of the full length protein can be used to identify turnover.

Further, the antibodies can be used to assess expression in disease states such as in active stages of the disease or in an individual with a predisposition toward disease related to the protein's function. When a disorder is caused by an inappropriate tissue distribution, developmental expression, level of expression of the protein, or expressed/processed form, the antibody can be prepared against the normal protein. Experimental data as provided in FIG. 1 indicates expression in humans in placenta choriocarcinomas, ovary adenocarcinomas, retinoblastomas of the eye, brain neuroblastomas, endometrium adenocarcinomas, colon, lung small cell carcinomas, T-lymphocytes, ovarian tumors, pheochromocytomas, fetal liver/spleen, Burkitt's lymphoma, and leukocytes. If a disorder is characterized by a specific mutation in the protein, antibodies specific for this mutant protein can be used to assay for the presence of the specific mutant protein.

The antibodies can also be used to assess normal and aberrant subcellular localization of cells in the various tissues in an organism. Experimental data as provided in FIG. 1 indicates expression in humans in placenta choriocarcinomas, ovary adenocarcinomas, retinoblastomas of the eye, brain neuroblastomas, endometrium adenocarcinomas, colon, lung small cell carcinomas, T-lymphocytes, ovarian tumors, pheochromocytomas, fetal liver/spleen, Burkitt's lymphoma, and leukocytes. The diagnostic uses can be applied, not only in genetic testing, but also in monitoring a treatment modality. Accordingly, where treatment is ultimately aimed at correcting expression level or the presence of aberrant sequence and aberrant tissue distribution or developmental expression, antibodies directed against the protein or relevant fragments can be used to monitor therapeutic efficacy.

Additionally, antibodies are useful in pharmacogenomic analysis. Thus, antibodies prepared against polymorphic proteins can be used to identify individuals that require modified treatment modalities. The antibodies are also useful as diagnostic tools as an immunological marker for aberrant protein analyzed by electrophoretic mobility, isoelectric point, tryptic peptide digest, and other physical assays known to those in the art.

The antibodies are also useful for tissue typing. Experimental data as provided in FIG. 1 indicates expression in humans in placenta choriocarcinomas, ovary adenocarcinomas, retinoblastomas of the eye, brain neuroblastomas, endometrium adenocarcinomas, colon, lung small cell carcinomas, T-lymphocytes, ovarian tumors, pheochromocytomas, fetal liver/spleen, Burkitt's lymphoma, and leukocytes. Thus, where a specific protein has been correlated with expression in a specific tissue, antibodies that are specific for this protein can be used to identify a tissue type.

The antibodies are also useful for inhibiting protein function, for example, blocking the binding of the transporter cofactor peptide to a binding partner such as a ligand or protein binding partner. These uses can also be applied in a therapeutic context in which treatment involves inhibiting the protein's function. An antibody can be used, for example, to block binding, thus modulating (agonizing or antagonizing) the peptides activity. Antibodies can be prepared against specific fragments containing sites required for function or against intact protein that is associated with a cell or cell membrane. See FIG. 2 for structural information relating to the proteins of the present invention.

The invention also encompasses kits for using antibodies to detect the presence of a protein in a biological sample. The kit can comprise antibodies such as a labeled or labelable antibody and a compound or agent for detecting protein in a biological sample; means for determining the amount of protein in the sample; means for comparing the amount of protein in the sample with a standard; and instructions for use. Such a kit can be supplied to detect a single protein or epitope or can be configured to detect one of a multitude of epitopes, such as in an antibody detection array. Arrays are described in detail below for nucleic acid arrays and similar methods have been developed for antibody arrays.

Nucleic Acid Molecules The present invention further provides isolated nucleic acid molecules that encode a transporter cofactor peptide or protein of the present invention (cDNA, transcript and genomic sequence). Such nucleic acid molecules will consist of, consist essentially of, or comprise a nucleotide sequence that encodes one of the transporter cofactor peptides of the present invention, an allelic variant thereof, or an ortholog or paralog thereof.

As used herein, an “isolated” nucleic acid molecule is one that is separated from other nucleic acid present in the natural source of the nucleic acid. Preferably, an “isolated” nucleic acid is free of sequences that naturally flank the nucleic acid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. However, there can be some flanking nucleotide sequences, for example up to about 5 KB, 4 KB, 3 KB, 2 KB, or 1 KB or less, particularly contiguous peptide encoding sequences and peptide encoding sequences within the same gene but separated by introns in the genomic sequence. The important point is that the nucleic acid is isolated from remote and unimportant flanking sequences such that it can be subjected to the specific manipulations described herein such as recombinant expression, preparation of probes and primers, and other uses specific to the nucleic acid sequences.

Moreover, an “isolated” nucleic acid molecule, such as a transcript/cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. However, the nucleic acid molecule can be fused to other coding or regulatory sequences and still be considered isolated.

For example, recombinant DNA molecules contained in a vector are considered isolated. Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the isolated DNA molecules of the present invention. Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically.

Accordingly, the present invention provides nucleic acid molecules that consist of the nucleotide sequence shown in FIG. 1 or 3 (SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic sequence), or any nucleic acid molecule that encodes the protein provided in FIG. 2, SEQ ID NO:2. A nucleic acid molecule consists of a nucleotide sequence when the nucleotide sequence is the complete nucleotide sequence of the nucleic acid molecule.

The present invention further provides nucleic acid molecules that consist essentially of the nucleotide sequence shown in FIG. 1 or 3 (SEQ ID NO: 1, transcript sequence and SEQ ID NO:3, genomic sequence), or any nucleic acid molecule that encodes the protein provided in FIG. 2, SEQ ID NO:2. A nucleic acid molecule consists essentially of a nucleotide sequence when such a nucleotide sequence is present with only a few additional nucleic acid residues in the final nucleic acid molecule.

The present invention further provides nucleic acid molecules that comprise the nucleotide sequences shown in FIG. 1 or 3 (SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic sequence), or any nucleic acid molecule that encodes the protein provided in FIG. 2, SEQ ID NO:2. A nucleic acid molecule comprises a nucleotide sequence when the nucleotide sequence is at least part of the final nucleotide sequence of the nucleic acid molecule. In such a fashion, the nucleic acid molecule can be only the nucleotide sequence or have additional nucleic acid residues, such as nucleic acid residues that are naturally associated with it or heterologous nucleotide sequences. Such a nucleic acid-molecule can have a few additional nucleotides or can comprise several hundred or more additional nucleotides. A brief description of how various types of these nucleic acid molecules can be readily made/isolated is provided below.

In FIGS. 1 and 3, both coding and non-coding sequences are provided. Because of the source of the present invention, humans genomic sequence (FIG. 3) and cDNA/transcript sequences (FIG. 1), the nucleic acid molecules in the Figures will contain genomic intronic sequences, 5′ and 3′ non-coding sequences, gene regulatory regions and non-coding intergenic sequences. In general such sequence features are either noted in FIGS. 1 and 3 or can readily be identified using computational tools known in the art. As discussed below, some of the non-coding regions, particularly gene regulatory elements such as promoters, are useful for a variety of purposes, e.g. control of heterologous gene expression, target for identifying gene activity modulating compounds, and are particularly claimed as fragments of the genomic sequence provided herein.

The isolated nucleic acid molecules can encode the mature protein plus additional amino or carboxyl-terminal amino acids, or amino acids interior to the mature peptide (when the mature form has more than one peptide chain, for instance). Such sequences may play a role in processing of a protein from precursor to a mature form, facilitate protein trafficking, prolong or shorten protein half-life or facilitate manipulation of a protein for assay or production, among other things. As generally is the case in situ, the additional amino acids may be processed away from the mature protein by cellular enzymes.

As mentioned above, the isolated nucleic acid molecules include, but are not limited to, the sequence encoding the transporter cofactor peptide alone, the sequence encoding the mature peptide and additional coding sequences, such as a leader or secretory sequence (e.g., a pre-pro or pro-protein sequence), the sequence encoding the mature peptide, with or without the additional coding sequences, plus additional non-coding sequences, for example introns and non-coding 5′ and 3′ sequences such as transcribed but non-translated sequences that play a role in transcription, mRNA processing (including splicing and polyadenylation signals), ribosome binding and stability of mRNA. In addition, the nucleic acid molecule may be fused to a marker sequence encoding, for example, a peptide that facilitates purification.

Isolated nucleic acid molecules can be in the form of RNA, such as mRNA, or in the form DNA, including cDNA and genomic DNA obtained by cloning or produced by chemical synthetic techniques or by a combination thereof. The nucleic acid, especially DNA, can be double-stranded or single-stranded. Single-stranded nucleic acid can be the coding strand (sense strand) or the non-coding strand (anti-sense strand).

The invention further provides nucleic acid molecules that encode fragments of the peptides of the present invention as well as nucleic acid molecules that encode obvious variants of the transporter cofactor proteins of the present invention that are described above. Such nucleic acid molecules may be naturally occurring, such as allelic variants (same locus), paralogs (different locus), and orthologs (different organism), or may be constructed by recombinant DNA methods or by chemical synthesis. Such non-naturally occurring variants may be made by mutagenesis techniques, including those applied to nucleic acid molecules, cells, or organisms. Accordingly, as discussed above, the variants can contain nucleotide substitutions, deletions, inversions and insertions. Variation can occur in either or both the coding and non-coding regions. The variations can produce both conservative and non-conservative amino acid substitutions.

The present invention further provides non-coding fragments of the nucleic acid molecules provided in FIGS. 1 and 3. Preferred non-coding fragments include, but are not limited to, promoter sequences, enhancer sequences, gene modulating sequences and gene termination sequences. Such fragments are useful in controlling heterologous gene expression and in developing screens to identify gene-modulating agents. A promoter can readily be identified as being 5′ to the ATG start site in the genomic sequence provided in FIG. 3.

A fragment comprises a contiguous nucleotide sequence greater than 12 or more nucleotides. Further, a fragment could at least 30, 40, 50, 100, 250 or 500 nucleotides in length. The length of the fragment will be based on its intended use. For example, the fragment can encode epitope bearing regions of the peptide, or can be useful as DNA probes and primers. Such fragments can be isolated using the known nucleotide sequence to synthesize an oligonucleotide probe. A labeled probe can then be used to screen a cDNA library, genomic DNA library, or mRNA to isolate nucleic acid corresponding to the coding region. Further, primers can be used in PCR reactions to clone specific regions of gene.

A probe/primer typically comprises substantially a purified oligonucleotide or oligonucleotide pair. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, 20, 25, 40, 50 or more consecutive nucleotides.

Orthologs, homologs, and allelic variants can be identified using methods well known in the art. As described in the Peptide Section, these variants comprise a nucleotide sequence encoding a peptide that is typically 60-70%, 70-80%, 80-90%, and more typically at least about 90-95% or more homologous to the nucleotide sequence shown in the Figure sheets or a fragment of this sequence. Such nucleic acid molecules can readily be identified as being able to hybridize under moderate to stringent conditions, to the nucleotide sequence shown in the Figure sheets or a fragment of the sequence. Allelic variants can readily be determined by genetic locus of the encoding gene. The gene encoding the novel transporter regulatory factor protein of the present invention is located on a genome component that has been mapped to human chromosome 17 (as indicated in FIG. 3), which is supported by multiple lines of evidence, such as STS and BAC map data.

FIG. 3 provides information on SNPs that have been found in the gene encoding the transporter cofactor protein of the present invention. SNPs were identified at 18 different nucleotide positions. Changes in the amino acid sequence caused by these SNPs can readily be determined using the universal genetic code and the protein sequence provided in FIG. 2 as a reference. SNPs outside the ORF and in introns may affect control/regulatory elements. Positioning of each SNP in exons, introns, or outside the ORF can readily be determined using the DNA positions given for each SNP and the start/stop, exon, and intron genomic coordinates given in FIG. 3.

As used herein, the term “hybridizes under stringent conditions” is intended to describe conditions for hybridization and washing under which nucleotide sequences encoding a peptide at least 60-70% homologous to each other typically remain hybridized to each other. The conditions can be such that sequences at least about 60%, at least about 70%, or at least about 80% or more homologous to each other typically remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. One example of stringent hybridization conditions are hybridization in 6× sodium chloride/sodium citrate (SSC) at about 45C, followed by one or more washes in 0.2×SSC, 0.1% SDS at 50-65C. Examples of moderate to low stringency hybridization conditions are well known in the art.

Nucleic Acid Molecule Uses

The nucleic acid molecules of the present invention are useful for probes, primers, chemical intermediates, and in biological assays. The nucleic acid molecules are useful as a hybridization probe for messenger RNA, transcript/cDNA and genomic DNA to isolate full-length cDNA and genomic clones encoding the peptide described in FIG. 2 and to isolate cDNA and genomic clones that correspond to variants (alleles, orthologs, etc.) producing the same or related peptides shown in FIG. 2. As illustrated in FIG. 3, SNPs were identified at 18 different nucleotide positions.

The probe can correspond to any sequence along the entire length of the nucleic acid molecules provided in the Figures. Accordingly, it could be derived from 5′ noncoding regions, the coding region, and 3′ noncoding regions. However, as discussed, fragments are not to be construed as encompassing fragments disclosed prior to the present invention.

The nucleic acid molecules are also useful as primers for PCR to amplify any given region of a nucleic acid molecule and are useful to synthesize antisense molecules of desired length and sequence.

The nucleic acid molecules are also useful for constructing recombinant vectors. Such vectors include expression vectors that express a portion of, or all of, the peptide sequences. Vectors also include insertion vectors, used to integrate into another nucleic acid molecule sequence, such as into the cellular genome, to alter in situ expression of a gene and/or gene product. For example, an endogenous coding sequence can be replaced via homologous recombination with all or part of the coding region containing one or more specifically introduced mutations.

The nucleic acid molecules are also useful for expressing antigenic portions of the proteins.

The nucleic acid molecules are also useful as probes for determining the chromosomal positions of the nucleic acid molecules by means of in situ hybridization methods. The gene encoding the novel transporter regulatory factor protein of the present invention is located on a genome component that has been mapped to human chromosome 17 (as indicated in FIG. 3), which is supported by multiple lines of evidence, such as STS and BAC map data.

The nucleic acid molecules are also useful in making vectors containing the gene regulatory regions of the nucleic acid molecules of the present invention.

The nucleic acid molecules are also useful for designing ribozymes corresponding to all, or a part, of the mRNA produced from the nucleic acid molecules described herein.

The nucleic acid molecules are also useful for making vectors that express part, or all, of the peptides.

The nucleic acid molecules are also useful for constructing host cells expressing a part, or all, of the nucleic acid molecules and peptides.

The nucleic acid molecules are also useful for constructing transgenic animals expressing all, or a part, of the nucleic acid molecules and peptides.

The nucleic acid molecules are also useful as hybridization probes for determining the presence, level, form and distribution of nucleic acid expression. Experimental data as provided in FIG. 1 indicates that the transporter cofactor proteins of the present invention are expressed in humans in placenta choriocarcinomas, ovary adenocarcinomas, retinoblastomas of the eye, brain neuroblastomas, endometrium adenocarcinomas, colon, lung small cell carcinomas, T-lymphocytes, ovarian tumors, pheochromocytomas, fetal liver/spleen, and Burkitt's lymphoma, as indicated by virtual northern blot analysis. In addition, PCR-based tissue screening panels indicate expression in human leukocytes.

Accordingly, the probes can be used to detect the presence of, or to determine levels of, a specific nucleic acid molecule in cells, tissues, and in organisms. The nucleic acid whose level is determined can be DNA or RNA. Accordingly, probes corresponding to the peptides described herein can be used to assess expression and/or gene copy number in a given cell, tissue, or organism. These uses are relevant for diagnosis of disorders involving an increase or decrease in transporter cofactor protein expression relative to normal results.

In vitro techniques for detection of mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detecting DNA include Southern hybridizations and in situ hybridization.

Probes can be used as a part of a diagnostic test kit for identifying cells or tissues that express a transporter cofactor protein, such as by measuring a level of a transporter cofactor-encoding nucleic acid in a sample of cells from a subject e.g., mRNA or genomic DNA, or determining if a transporter cofactor gene has been mutated. Experimental data as provided in FIG. 1 indicates that the transporter cofactor proteins of the present invention are expressed in humans in placenta choriocarcinomas, ovary adenocarcinomas, retinoblastomas of the eye, brain neuroblastomas, endometrium adenocarcinomas, colon, lung small cell carcinomas, T-lymphocytes, ovarian tumors, pheochromocytomas, fetal liver/spleen, and Burkitt's lymphoma, as indicated by virtual northern blot analysis. In addition, PCR-based tissue screening panels indicate expression in human leukocytes.

Nucleic acid expression assays are useful for drug screening to identify compounds that modulate transporter cofactor nucleic acid expression.

The invention thus provides a method for identifying a compound that can be used to treat a disorder associated with nucleic acid expression of the transporter cofactor gene, particularly biological and pathological processes that are mediated by the transporter cofactor in cells and tissues that express it. Experimental data as provided in FIG. 1 indicates expression in humans in placenta choriocarcinomas, ovary adenocarcinomas, retinoblastomas of the eye, brain neuroblastomas, endometrium adenocarcinomas, colon, lung small cell carcinomas, T-lymphocytes, ovarian tumors, pheochromocytomas, fetal liver/spleen, Burkitt's lymphoma, and leukocytes. The method typically includes assaying the ability of the compound to modulate the expression of the transporter cofactor nucleic acid and thus identifying a compound that can be used to treat a disorder characterized by undesired transporter cofactor nucleic acid expression. The assays can be performed in cell-based and cell-free systems. Cell-based assays include cells naturally expressing the transporter cofactor nucleic acid or recombinant cells genetically engineered to express specific nucleic acid sequences.

The assay for transporter cofactor nucleic acid expression can involve direct assay of nucleic acid levels, such as mRNA levels, or on collateral compounds involved in the signal pathway. Further, the expression of genes that are up- or down-regulated in response to the transporter cofactor signal pathway can also be assayed. In this embodiment the regulatory regions of these genes can be operably linked to a reporter gene such as luciferase.

Thus, modulators of transporter cofactor gene expression can be identified in a method wherein a cell is contacted with a candidate compound and the expression of mRNA determined. The level of expression of transporter cofactor mRNA in the presence of the candidate compound is compared to the level of expression of transporter cofactor mRNA in the absence of the candidate compound. The candidate compound can then be identified as a modulator of nucleic acid expression based on this comparison and be used, for example to treat a disorder characterized by aberrant nucleic acid expression. When expression of mRNA is statistically significantly greater in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of nucleic acid expression. When nucleic acid expression is statistically significantly less in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of nucleic acid expression.

The invention further provides methods of treatment, with the nucleic acid as a target, using a compound identified through drug screening as a gene modulator to modulate transporter cofactor nucleic acid expression in cells and tissues that express the transporter cofactor. Experimental data as provided in FIG. 1 indicates that the transporter cofactor proteins of the present invention are expressed in humans in placenta choriocarcinomas, ovary adenocarcinomas, retinoblastomas of the eye, brain neuroblastomas, endometrium adenocarcinomas, colon, lung small cell carcinomas, T-lymphocytes, ovarian tumors, pheochromocytomas, fetal liver/spleen, and Burkitt's lymphoma, as indicated by virtual northern blot analysis. In addition, PCR-based tissue screening panels indicate expression in human leukocytes. Modulation includes both up-regulation (i.e. activation or agonization) or down-regulation (suppression or antagonization) or nucleic acid expression.

Alternatively, a modulator for transporter cofactor nucleic acid expression can be a small molecule or drug identified using the screening assays described herein as long as the drug or small molecule inhibits the transporter cofactor nucleic acid expression in the cells and tissues that express the protein. Experimental data as provided in FIG. 1 indicates expression in humans in placenta choriocarcinomas, ovary adenocarcinomas, retinoblastomas of the eye, brain neuroblastomas, endometrium adenocarcinomas, colon, lung small cell carcinomas, T-lymphocytes, ovarian tumors, pheochromocytomas, fetal liver/spleen, Burkitt's lymphoma, and leukocytes.

The nucleic acid molecules are also useful for monitoring the effectiveness of modulating compounds on the expression or activity of the transporter cofactor gene in clinical trials or in a treatment regimen. Thus, the gene expression pattern can serve as a barometer for the continuing effectiveness of treatment with the compound, particularly with compounds to which a patient can develop resistance. The gene expression pattern can also serve as a marker indicative of a physiological response of the affected cells to the compound. Accordingly, such monitoring would allow either increased administration of the compound or the administration of alternative compounds to which the patient has not become resistant. Similarly, if the level of nucleic acid expression falls below a desirable level, administration of the compound could be commensurately decreased.

The nucleic acid molecules are also useful in diagnostic assays for qualitative changes in transporter cofactor nucleic acid expression, and particularly in qualitative changes that lead to pathology. The nucleic acid molecules can be used to detect mutations in transporter cofactor genes and gene expression products such as mRNA. The nucleic acid molecules can be used as hybridization probes to detect naturally occurring genetic mutations in the transporter cofactor gene and thereby to determine whether a subject with the mutation is at risk for a disorder caused by the mutation. Mutations include deletion, addition, or substitution of one or more nucleotides in the gene, chromosomal rearrangement, such as inversion or transposition, modification of genomic DNA, such as aberrant methylation patterns or changes in gene copy number, such as amplification. Detection of a mutated form of the transporter cofactor gene associated with a dysfunction provides a diagnostic tool for an active disease or susceptibility to disease when the disease results from overexpression, underexpression, or altered expression of a transporter cofactor.

Individuals carrying mutations in the transporter cofactor gene can be detected at the nucleic acid level by a variety of techniques. FIG. 3 provides information on SNPs that have been found in the gene encoding the transporter cofactor of the present invention. SNPs were identified at 18 different nucleotide positions. Changes in the amino acid sequence caused by these SNPs can readily be determined using the universal genetic code and the protein sequence provided in FIG. 2 as a reference. SNPs outside the ORF and in introns may affect control/regulatory elements. Positioning of each SNP in exons, introns, or outside the ORF can readily be determined using the DNA positions given for each SNP and the start/stop, exon, and intron genomic coordinates given in FIG. 3. The gene encoding the novel transporter regulatory factor protein of the present invention is located on a genome component that has been mapped to human chromosome 17 (as indicated in FIG. 3), which is supported by multiple lines of evidence, such as STS and BAC map data. Genomic DNA can be analyzed directly or can be amplified by using PCR prior to analysis. RNA or cDNA can be used in the same way. In some uses, detection of the mutation involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g. U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al., Science 241:1077-1080 (1988); and Nakazawa et al., PNAS91:360-364 (1994)), the latter of which can be particularly useful for detecting point mutations in the gene (see Abravaya et al., Nucleic Acids Res. 23:675-682 (1995)). This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to a gene under conditions such that hybridization and amplification of the gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. Deletions and insertions can be detected by a change in size of the amplified product compared to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to normal RNA or antisense DNA sequences.

Alternatively, mutations in a transporter cofactor gene can be directly identified, for example, by alterations in restriction enzyme digestion patterns determined by gel electrophoresis.

Further, sequence-specific ribozymes (U.S. Pat. No. 5,498,531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site. Perfectly matched sequences can be distinguished from mismatched sequences by nuclease cleavage digestion assays or by differences in melting temperature.

Sequence changes at specific locations can also be assessed by nuclease protection assays such as RNase and S1 protection or the chemical cleavage method. Furthermore, sequence differences between a mutant transporter cofactor gene and a wild-type gene

can be determined by direct DNA sequencing. A variety of automated sequencing procedures can be utilized when performing the diagnostic assays (Naeve, C. W., (1995) Biotechniques 19:448), including sequencing by mass spectrometry (see, e.g., PCT International Publication No. WO 94/16101; Cohen et al., Adv. Chromatogr. 36:127-162 (1996); and Griffin et al., Appl. Biochem. Biotechnol. 38:147-159 (1993)).

Other methods for detecting mutations in the gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA duplexes (Myers et al., Science 230:1242 (1985)); Cotton et al., PNAS 85:4397 (1988); Saleeba et al., Meth. EnzymoL 217:286-295 (1992)), electrophoretic mobility of mutant and wild type nucleic acid is compared (Orita et al., PNAS 86:2766 (1989); Cotton et al., Mutat. Res. 285:125-144 (1993); and Hayashi et al., Genet. Anal Tech. Appl. 9:73-79 (1992)), and movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (Myers et al., Nature 313:495 (1985)). Examples of other techniques for detecting point mutations include selective oligonucleotide hybridization, selective amplification, and selective primer extension.

The nucleic acid molecules are also useful for testing an individual for a genotype that while not necessarily causing the disease, nevertheless affects the treatment modality. Thus, the nucleic acid molecules can be used to study the relationship between an individual's genotype and the individual's response to a compound used for treatment (pharmacogenomic relationship). Accordingly, the nucleic acid molecules described herein can be used to assess the mutation content of the transporter cofactor gene in an individual in order to select an appropriate compound or dosage regimen for treatment. FIG. 3 provides information on SNPs that have been found in the gene encoding the transporter cofactor protein of the present invention. SNPs were identified at 18 different nucleotide positions. Changes in the amino acid sequence caused by these SNPs can readily be determined using the universal genetic code and the protein sequence provided in FIG. 2 as a reference. SNPs outside the ORF and in introns may affect control/regulatory elements. Positioning of each SNP in exons, introns, or outside the ORF can readily be determined using the DNA positions given for each SNP and the start/stop, exon, and intron genomic coordinates given in FIG. 3.

Thus nucleic acid molecules displaying genetic variations that affect treatment provide a diagnostic target that can be used to tailor treatment in an individual. Accordingly, the production of recombinant cells and animals containing these polymorphisms allow effective clinical design of treatment compounds and dosage regimens.

The nucleic acid molecules are thus useful as antisense constructs to control transporter cofactor gene expression in cells, tissues, and organisms. A DNA antisense nucleic acid molecule is designed to be complementary to a region of the gene involved in transcription, preventing transcription and hence production of transporter cofactor protein. An antisense RNA or DNA nucleic acid molecule would hybridize to the mRNA and thus block translation of mRNA into transporter cofactor protein.

Alternatively, a class of antisense molecules can be used to inactivate mRNA in order to decrease expression of transporter cofactor nucleic acid. Accordingly, these molecules can treat a disorder characterized by abnormal or undesired transporter cofactor nucleic acid expression. This technique involves cleavage by means of ribozymes containing nucleotide sequences complementary to one or more regions in the mRNA that attenuate the ability of the mRNA to be translated. Possible regions include coding regions and particularly coding regions corresponding to the catalytic and other functional activities of the transporter cofactor protein, such as ligand binding.

The nucleic acid molecules also provide vectors for gene therapy in patients containing cells that are aberrant in transporter cofactor gene expression. Thus, recombinant cells, which include the patient's cells that have been engineered ex vivo and returned to the patient, are introduced into an individual where the cells produce the desired transporter cofactor protein to treat the individual.

The invention also encompasses kits for detecting the presence of a transporter cofactor nucleic acid in a biological sample. Experimental data as provided in FIG. 1 indicates that the transporter cofactor proteins of the present invention are expressed in humans in placenta choriocarcinomas, ovary adenocarcinomas, retinoblastomas of the eye, brain neuroblastomas, endometrium adenocarcinomas, colon, lung small cell carcinomas, T-lymphocytes, ovarian tumors, pheochromocytomas, fetal liver/spleen, and Burkitt's lymphoma, as indicated by virtual northern blot analysis. In addition, PCR-based tissue screening panels indicate expression in human leukocytes. For example, the kit can comprise reagents such as a labeled or labelable nucleic acid or agent capable of detecting transporter cofactor nucleic acid in a biological sample; means for determining the amount of transporter cofactor nucleic acid in the sample; and means for comparing the amount of transporter cofactor nucleic acid in the sample with a standard. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect transporter cofactor protein mRNA or DNA.

Nucleic Acid Arrays

The present invention further provides nucleic acid detection kits, such as arrays or microarrays of nucleic acid molecules that are based on the sequence information provided in FIGS. 1 and 3 (SEQ ID NOS:1 and 3).

As used herein “Arrays” or “Microarrays” refers to an array of distinct polynucleotides or oligonucleotides synthesized on a substrate, such as paper, nylon or other type of membrane, filter, chip, glass slide, or any other suitable solid support. In one embodiment, the microarray is prepared and used according to the methods described in U.S. Pat. No. 5,837,832, Chee et al., PCT application W095/11995 (Chee et al.), Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) and Schena, M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all of which are incorporated herein in their entirety by reference. In other embodiments, such arrays are produced by the methods described by Brown et al., U.S. Pat. No. 5,807,522.

The microarray or detection kit is preferably composed of a large number of unique, single-stranded nucleic acid sequences, usually either synthetic antisense oligonucleotides or fragments of cDNAs, fixed to a solid support. The oligonucleotides are preferably about 6-60 nucleotides in length, more preferably 15-30 nucleotides in length, and most preferably about 20-25 nucleotides in length. For a certain type of microarray or detection kit, it may be preferable to use oligonucleotides that are only 7-20 nucleotides in length. The microarray or detection kit may contain oligonucleotides that cover the known 5′, or 3′, sequence, sequential oligonucleotides that cover the full length sequence; or unique oligonucleotides selected from particular areas along the length of the sequence. Polynucleotides used in the microarray or detection kit may be oligonucleotides that are specific to a gene or genes of interest.

In order to produce oligonucleotides to a known sequence for a microarray or detection kit, the gene(s) of interest (or an ORF identified from the contigs of the present invention) is typically examined using a computer algorithm which starts at the 5′ or at the 3′ end of the nucleotide sequence. Typical algorithms will then identify oligomers of defined length that are unique to the gene, have a GC content within a range suitable for hybridization, and lack predicted secondary structure that may interfere with hybridization. In certain situations it may be appropriate to use pairs of oligonucleotides on a microarray or detection kit. The “pairs” will be identical, except for one nucleotide that preferably is located in the center of the sequence. The second oligonucleotide in the pair (mismatched by one) serves as a control. The number of oligonucleotide pairs may range from two to one million. The oligomers are synthesized at designated areas on a substrate using a light-directed chemical process. The substrate may be paper, nylon or other type of membrane, filter, chip, glass slide or any other suitable solid support.

In another aspect, an oligonucleotide may be synthesized on the surface of the substrate by using a chemical coupling procedure and an ink jet application apparatus, as described in PCT application W095/251116 (Baldeschweiler et al.) which is incorporated herein in its entirety by reference. In another aspect, a “gridded” array analogous to a dot (or slot) blot may be used to arrange and link cDNA fragments or oligonucleotides to the surface of a substrate using a vacuum system, thermal, UV, mechanical or chemical bonding procedures. An array, such as those described above, may be produced by hand or by using available devices (slot blot or dot blot apparatus), materials (any suitable solid support), and machines (including robotic instruments), and may contain 8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or any other number be million which lends itself to the efficient use of commercially available instrumentation.

In order to conduct sample analysis using a microarray or detection kit, the RNA or DNA from a biological sample is made into hybridization probes. The mRNA is isolated, and cDNA is produced and used as a template to make antisense RNA (aRNA). The aRNA is amplified in the presence of fluorescent nucleotides, and labeled probes are incubated with the microarray or detection kit so that the probe sequences hybridize to complementary oligonucleotides of the microarray or detection kit. Incubation conditions are adjusted so that hybridization occurs with precise complementary matches or with various degrees of less complementarity. After removal of nonhybridized probes, a scanner is used to determine the levels and patterns of fluorescence. The scanned images are examined to determine degree of complementarity and the relative abundance of each oligonucleotide sequence on the microarray or detection kit. The biological samples may be obtained from any bodily fluids (such as blood, urine, saliva, phlegm, gastric juices, etc.), cultured cells, biopsies, or other tissue preparations. A detection system may be used to measure the absence, presence, and amount of hybridization for all of the distinct sequences simultaneously. This data may be used for large-scale correlation studies on the sequences, expression patterns, mutations, variants, or polymorphisms among samples.

Using such arrays, the present invention provides methods to identify the expression of the transporter cofactor proteins/peptides of the present invention. In detail, such methods comprise incubating a test sample with one or more nucleic acid molecules and assaying for binding of the nucleic acid molecule with components within the test sample. Such assays will typically involve arrays comprising many genes, at least one of which is a gene of the present invention and or alleles of the transporter cofactor gene of the present invention. FIG. 3 provides information on SNPs that have been found in the gene encoding the transporter cofactor protein of the present invention. SNPs were identified at 18 different nucleotide positions. Changes in the amino acid sequence caused by these SNPs can readily be determined using the universal genetic code and the protein sequence provided in FIG. 2 as a reference. SNPs outside the ORF and in introns may affect control/regulatory elements. Positioning of each SNP in exons, introns, or outside the ORF can readily be determined using the DNA positions given for each SNP and the start/stop, exon, and intron genomic coordinates given in FIG. 3.

Conditions for incubating a nucleic acid molecule with a test sample vary. Incubation conditions depend on the format employed in the assay, the detection methods employed, and the type and nature of the nucleic acid molecule used in the assay. One skilled in the art will recognize that any one of the commonly available hybridization, amplification or array assay formats can readily be adapted to employ the novel fragments of the Human genome disclosed herein. Examples of such assays can be found in Chard, T, An Introduction to Radioimmunoassay and Related Techniques, Elsevier Science Publishers, Amsterdam, The Netherlands (1986); Bullock, G. R. et al., Techniques in Immunocytochemistry, Academic Press, Orlando, FL Vol. 1 (1982), Vol. 2 (1983), Vol.3 (1985); Tijssen, P., Practice and Theory of Enzyme Immunoassays: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985).

The test samples of the present invention include cells, protein or membrane extracts of cells. The test sample used in the above-described method will vary based on the assay format, nature of the detection method and the tissues, cells or extracts used as the sample to be assayed. Methods for preparing nucleic acid extracts or of cells are well known in the art and can be readily be adapted in order to obtain a sample that is compatible with the system utilized.

In another embodiment of the present invention, kits are provided which contain the necessary reagents to carry out the assays of the present invention.

Specifically, the invention provides a compartmentalized kit to receive, in close confinement, one or more containers which comprises: (a) a first container comprising one of the nucleic acid molecules that can bind to a fragment of the Human genome disclosed herein; and (b) one or more other containers comprising one or more of the following: wash reagents, reagents capable of detecting presence of a bound nucleic acid.

In detail, a compartmentalized kit includes any kit in which reagents are contained in separate containers. Such containers include small glass containers, plastic containers, strips of plastic, glass or paper, or arraying material such as silica. Such containers allows one to efficiently transfer reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated, and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another. Such containers will include a container which will accept the test sample, a container which contains the nucleic acid probe, containers which contain wash reagents (such as phosphate buffered saline, Tris-buffers, etc.), and containers which contain the reagents used to detect the bound probe. One skilled in the art will readily recognize that the previously unidentified transporter cofactor gene of the present invention can be routinely identified using the sequence information disclosed herein can be readily incorporated into one of the established kit formats which are well known in the art, particularly expression arrays.

Vectors/host cells

The invention also provides vectors containing the nucleic acid molecules described herein. The term “vector” refers to a vehicle, preferably a nucleic acid molecule, which can transport the nucleic acid molecules. When the vector is a nucleic acid molecule, the nucleic acid molecules are covalently linked to the vector nucleic acid. With this aspect of the invention, the vector includes a plasmid, single or double stranded phage, a single or double stranded RNA or DNA viral vector, or artificial chromosome, such as a BAC, PAC, YAC, OR MAC.

A vector can be maintained in the host cell as an extrachromosomal element where it replicates and produces additional copies of the nucleic acid molecules. Alternatively, the vector may integrate into the host cell genome and produce additional copies of the nucleic acid molecules when the host cell replicates.

The invention provides vectors for the maintenance (cloning vectors) or vectors for expression (expression vectors) of the nucleic acid molecules. The vectors can function in procaryotic or eukaryotic cells or in both (shuttle vectors).

Expression vectors contain cis-acting regulatory regions that are operably linked in the vector to the nucleic acid molecules such that transcription of the nucleic acid molecules is allowed in a host cell. The nucleic acid molecules can be introduced into the host cell with a separate nucleic acid molecule capable of affecting transcription. Thus, the second nucleic acid molecule may provide a trans-acting factor interacting with the cis-regulatory control region to allow transcription of the nucleic acid molecules from the vector. Alternatively, a trans-acting factor may be supplied by the host cell. Finally, a trans-acting factor can be produced from the vector itself It is understood, however, that in some embodiments, transcription and/or translation of the nucleic acid molecules can occur in a cell-free system.

The regulatory sequence to which the nucleic acid molecules described herein can be operably linked include promoters for directing mRNA transcription. These include, but are not limited to, the left promoter from bacteriophage λ, the lac, TRP, and TAC promoters from E. coli, the early and late promoters from SV40, the CMV immediate early promoter, the adenovirus early and late promoters, and retrovirus long-terminal repeats.

In addition to control regions that promote transcription, expression vectors may also include regions that modulate transcription, such as repressor binding sites and enhancers. Examples include the SV40 enhancer, the cytomegalovirus immediate early enhancer, polyoma enhancer, adenovirus enhancers, and retrovirus LTR enhancers.

In addition to containing sites for transcription initiation and control, expression vectors can also contain sequences necessary for transcription termination and, in the transcribed region a ribosome binding site for translation. Other regulatory control elements for expression include initiation and termination codons as well as polyadenylation signals. The person of ordinary skill in the art would be aware of the numerous regulatory sequences that are useful in expression vectors. Such regulatory sequences are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1989).

A variety of expression vectors can be used to express a nucleic acid molecule. Such vectors include chromosomal, episomal, and virus-derived vectors, for example vectors derived from bacterial plasmids, from bacteriophage, from yeast episomes, from yeast chromosomal elements, including yeast artificial chromosomes, from viruses such as baculoviruses, papovaviruses such as SV40, Vaccinia viruses, adenoviruses, poxviruses, pseudorabies viruses, and retroviruses. Vectors may also be derived from combinations of these sources such as those derived from plasmid and bacteriophage genetic elements, e.g. cosmids and phagemids. Appropriate cloning and expression vectors for prokaryotic and eukaryotic hosts are described in Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1989).

The regulatory sequence may provide constitutive expression in one or more host cells (i.e. tissue specific) or may provide for inducible expression in one or more cell types such as by temperature, nutrient additive, or exogenous factor such as a hormone or other ligand. A variety of vectors providing for constitutive and inducible expression in prokaryotic and eukaryotic hosts are well known to those of ordinary skill in the art.

The nucleic acid molecules can be inserted into the vector nucleic acid by well-known methodology. Generally, the DNA sequence that will ultimately be expressed is joined to an expression vector by cleaving the DNA sequence and the expression vector with one or more restriction enzymes and then ligating the fragments together. Procedures for restriction enzyme digestion and ligation are well known to those of ordinary skill in the art.

The vector containing the appropriate nucleic acid molecule can be introduced into an appropriate host cell for propagation or expression using well-known techniques. Bacterial cells include, but are not limited to, E. coli, Streptomyces, and Salmonella typhimurium. Eukaryotic cells include, but are not limited to, yeast, insect cells such as Drosophila, animal cells such as COS and CHO cells, and plant cells.

As described herein, it may be desirable to express the peptide as a fusion protein. Accordingly, the invention provides fusion vectors that allow for the production of the peptides. Fusion vectors can increase the expression of a recombinant protein, increase the solubility of the recombinant protein, and aid in the purification of the protein by acting for example as a ligand for affinity purification. A proteolytic cleavage site may be introduced at the junction of the fusion moiety so that the desired peptide can ultimately be separated from the fusion moiety. Proteolytic enzymes include, but are not limited to, factor Xa, thrombin, and enterotransporter. Typical fusion expression vectors include pGEX (Smith et al., Gene 67:31-40 (1988)), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein. Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al., Gene 69:301 315 (1988)) and pET 11d (Studier et al., Gene Expression Technology: Methods in Enzymology 185:60-89 (1990)).

Recombinant protein expression can be maximized in host bacteria by providing a genetic background wherein the host cell has an impaired capacity to proteolytically cleave the recombinant protein. (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 119-128). Alternatively, the sequence of the nucleic acid molecule of interest can be altered to provide preferential codon usage for a specific host cell, for example E coli. (Wada et al., Nucleic Acids Res. 20:2111-2118 (1992)).

The nucleic acid molecules can also be expressed by expression vectors that are operative in yeast. Examples of vectors for expression in yeast e.g., S. cerevisiae include pYepSec1 (Baldari, et al., EMBO J. 6:229-234 (1987)), pMFa (Kurjan et al., Cell 30:933-943(1982)), pJRY88 (Schultz et al., Gene 54:113-123 (1987)), and pYES2 (Invitrogen Corporation, San Diego, Calif.).

The nucleic acid molecules can also be expressed in insect cells using, for example, baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al., Mol. Cell Biol. 3:2156-2165 (1983)) and the pVL series (Lucklow et al., Virology 170:31-39 (1989)).

In certain embodiments of the invention, the nucleic acid molecules described herein are expressed in mammalian cells using mammalian expression vectors. Examples of mammalian expression vectors include pCDM8 (Seed, B. Nature 329:840(1987)) and pMT2PC (Kaufman et al., EMBO J. 6:187-195 (1987)).

The expression vectors listed herein are provided by way of example only of the well-known vectors available to those of ordinary skill in the art that would be useful to express the nucleic acid molecules. The person of ordinary skill in the art would be aware of other vectors suitable for maintenance propagation or expression of the nucleic acid molecules described herein. These are found for example in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

The invention also encompasses vectors in which the nucleic acid sequences described herein are cloned into the vector in reverse orientation, but operably linked to a regulatory sequence that permits transcription of anti sense RNA. Thus, an anti sense transcript can be produced to all, or to a portion, of the nucleic acid molecule sequences described herein, including both coding and non-coding regions. Expression of this antisense RNA is subject to each of the parameters described above in relation to expression of the sense RNA (regulatory sequences, constitutive or inducible expression, tissue-specific expression).

The invention also relates to recombinant host cells containing the vectors described herein. Host cells therefore include prokaryotic cells, lower eukaryotic cells such as yeast, other eukaryotic cells such as insect cells, and higher eukaryotic cells such as mammalian cells.

The recombinant host cells are prepared by introducing the vector constructs described herein into the cells by techniques readily available to the person of ordinary skill in the art. These include, but are not limited to, calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, lipofection, and other techniques such as those found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

Host cells can contain more than one vector. Thus, different nucleotide sequences can be introduced on different vectors of the same cell. Similarly, the nucleic acid molecules can be introduced either alone or with other nucleic acid molecules that are not related to the nucleic acid molecules such as those providing trans-acting factors for expression vectors. When more than one vector is introduced into a cell, the vectors can be introduced independently, co-introduced or joined to the nucleic acid molecule vector.

In the case of bacteriophage and viral vectors, these can be introduced into cells as packaged or encapsulated virus by standard procedures for infection and transduction. Viral vectors can be replication-competent or replication-defective. In the case in which viral replication is defective, replication will occur in host cells providing functions that complement the defects.

Vectors generally include selectable markers that enable the selection of the subpopulation of cells that contain the recombinant vector constructs. The marker can be contained in the same vector that contains the nucleic acid molecules described herein or may be on a separate vector. Markers include tetracycline or ampicillin-resistance genes for prokaryotic host cells and dihydrofolate reductase or neomycin resistance for eukaryotic host cells. However, any marker that provides selection for a phenotypic trait will be effective.

While the mature proteins can be produced in bacteria, yeast, mammalian cells, and other cells under the control of the appropriate regulatory sequences, cell-free transcription and translation systems can also be used to produce these proteins using RNA derived from the DNA constructs described herein.

Where secretion of the peptide is desired, which is difficult to achieve with multi-transmembrane domain containing proteins such as transporters, appropriate secretion signals are incorporated into the vector. The signal sequence can be endogenous to the peptides or heterologous to these peptides.

Where the peptide is not secreted into the medium, which is typically the case with transporter cofactors, the protein can be isolated from the host cell by standard disruption procedures, including freeze thaw, sonication, mechanical disruption, use of lysing agents and the like. The peptide can then be recovered and purified by well-known purification methods including ammonium sulfate precipitation, acid extraction, anion or cationic exchange chromatography, phosphocellulose chromatography, hydrophobic-interaction chromatography, affinity chromatography, hydroxylapatite chromatography, lectin chromatography, or high performance liquid chromatography.

It is also understood that depending upon the host cell in recombinant production of the peptides described herein, the peptides can have various glycosylation patterns, depending upon the cell, or maybe non-glycosylated as when produced in bacteria. In addition, the peptides may include an initial modified methionine in some cases as a result of a host-mediated process.

Uses of vectors and host cells

The recombinant host cells expressing the peptides described herein have a variety of uses. First, the cells are useful for producing a transporter cofactor protein or peptide that can be further purified to produce desired amounts of transporter cofactor protein or fragments. Thus, host cells containing expression vectors are useful for peptide production.

Host cells are also useful for conducting cell-based assays involving the transporter cofactor protein or transporter cofactor protein fragments, such as those described above as well as other formats known in the art. Thus, a recombinant host cell expressing a native transporter cofactor protein is useful for assaying compounds that stimulate or inhibit transporter cofactor function.

Host cells are also useful for identifying transporter cofactor mutants in which these functions are affected. If the mutants naturally occur and give rise to a pathology, host cells containing the mutations are useful to assay compounds that have a desired effect on the mutant transporter cofactor protein (for example, stimulating or inhibiting function) which may not be indicated by their effect on the native transporter cofactor protein.

Genetically engineered host cells can be further used to produce non-human transgenic animals. A transgenic animal is preferably a mammal, for example a rodent, such as a rat or mouse, in which one or more of the cells of the animal include a transgene. A transgene is exogenous DNA that is integrated into the genome of a cell from which a transgenic animal develops and which remains, in the genome of the mature animal in one or more cell types or tissues of the transgenic animal. These animals are useful for studying the function of a transporter cofactor and identifying and evaluating modulators of transporter cofactor activity. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, and amphibians.

A transgenic animal can be produced by introducing nucleic acid into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal. Any of the transporter cofactor nucleotide sequences can be introduced as a transgene into the genome of a non-human animal, such as a mouse.

Any of the regulatory or other sequences useful in expression vectors can form part of the transgenic sequence. This includes intronic sequences and polyadenylation signals, if not already included. A tissue-specific regulatory sequence(s) can be operably linked to the transgene to direct expression of the transporter cofactor to particular cells.

Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No. 4,873,191 by Wagner et al. and in Hogan, B., Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence of the transgene in its genome and/or expression of transgenic mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene can further be bred to other transgenic animals carrying other transgenes. A transgenic animal also includes animals in which the entire animal or tissues in the animal have been produced using the homologously recombinant host cells described herein.

In another embodiment, transgenic non-human animals can be produced which contain selected systems that allow for regulated expression of the transgene. One example of such a system is the cre/loxP recombinase system of bacteriophage P1. For a description of the cre/loxP recombinase system, see, e.g., Lakso et al. PNAS 89:6232-6236 (1992). Another example of a recombinase system is the FLP recombinase system of S. cerevisiae (O'Gorman et al. Science 251:1351-1355 (1991). If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein is required. Such animals can be provided through the construction of “double” transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.

Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, I. et al. Nature 385:810-813 (1997) and PCT International Publication Nos. WO 97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic cell, from the transgenic animal can be isolated and induced to exit the growth cycle and enter G₀ phase. The quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated. The reconstructed oocyte is then cultured such that it develops to morula or blastocyst and then transferred to pseudopregnant female foster animal. The offspring born of this female foster animal will be a clone of the animal from which the cell, e.g., the somatic cell, is isolated.

Transgenic animals containing recombinant cells that express the peptides described herein are useful to conduct the assays described herein in an in vivo context. Accordingly, the various physiological factors that are present in vivo and that could effect ligand binding, transporter cofactor activation, and signal transduction, may not be evident from in vitro cell-free or cell-based assays. Accordingly, it is useful to provide non-human transgenic animals to assay in vivo transporter cofactor function, including ligand interaction, the effect of specific mutatations on transporter cofactor function and ligand interaction, and the effect of chimeric transporter cofactors. It is also possible to assess the effect of null mutations, that is mutations that substantially or completely eliminate one or more transporter cofactor functions.

All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described modes for carrying out the invention which are obvious to those skilled in the field of molecular biology or related fields are intended to be within the scope of the following claims.

4 1 1949 DNA Human 1 gtctgtggtc ctctctcggc tcctcgcggc tcgcggcggc cgacggttcc tgggacacct 60 gcttgcttgg cccgtccggc ggctcagggc ttctctgctg cgctcccggt tcgctggacg 120 ggaagaaggg ctgggccgtc ccgtcccgtc cccatcggaa ccccaagtcg cgccgctgac 180 ccgtcgcagg gcgagatgag cgcggacgca gcggccgggg cgcccctgcc ccggctctgc 240 tgcctggaga agggtccgaa cggctacggc ttccacctgc acggggagaa gggcaagttg 300 ggccagtaca tccggctggt ggaggtgaac ggcgaaaacg tggagaagga gacccaccag 360 caggtggtga gccgcatccg cgccgcactc aacgccgtgc gcctgctggt ggtcgacccc 420 gagacggacg agcagctgca gaagctcggc gtccaggtcc gagaggagct gctgcgcgcc 480 caggaagcgc cggggcaggc cgagccgccg gccgccgccg aggtgcaggg ggctggcaac 540 gaaaatgagc ctcgcgaggc cgacaagagc cacccggagc agcgcgagct tcggcctcgg 600 ctctgtacca tgaagaaggg ccccagtggc tatggcttca acctgcacag cgacaagtcc 660 aagccaggcc agttcatccg gtcagtggac ccagactccc cggctgaggc ttcagggctc 720 cgggcccagg atcgcattgt ggaggtgaac ggggtctgca tggaggggaa gcagcatggg 780 gacgtggtgt ccgccatcag ggctggcggg gacgagacca agctgctggt ggtggacagg 840 gaaactgacg agttcttcaa gaaatgcaga gtgatcccat ctcaggagca cctgaatggt 900 cccctgcctg tgcccttcac caatggggag atacagaagg agaacagtcg tgaagccctg 960 gcagaggcag ccttggagag ccccaggcca gccctggtga gatccgcctc cagtgacacc 1020 agcgaggagc tgaattccca agacagcccc ccaaaacagg actccacagc gccctcgtct 1080 acctcctcct ccgaccccat cctagacttc aacatctccc tggccatggc caaagagagg 1140 gcccaccaga aacgcagcag caaacgggcc ccgcagatgg actggagcaa gaaaaacgaa 1200 ctcttcagca acctctgagc gccctgctgc cacccagtga ctggcagggc cgagccagca 1260 ttccacccca cctttttcct tctccccaat tactcccctg aatcaatgta caaatcagca 1320 cccacatccc ctttcttgac aaatgatttt tctagagaac tatgttcttc cctgacttta 1380 gggaaggtga atgtgttccc gtcctcccgc agtcagaaag gagactctgc ctccctcctc 1440 ctcactgagt gcctcatcct accgggtgtc cctttgccac cctgcctggg acatcgctgg 1500 aacctgcacc atgccaggat catgggacca ggcgagaggg caccctccct tcctccccca 1560 tgtgataaat gggtccaggg ctgatcaaag aactctgact gcagaactgc cgctctcagt 1620 ggacagggca tctgttaccc tgagacctgt ggcagacacg tcttgttttc atttgatttt 1680 tgttaagagt gcagtattgc agagtctaga ggaatttttg tttccttgat taacatgatt 1740 ttcctggttg ttacatccag ggcatggcag tggcctcagc cttaaacttt tgttcctact 1800 cccaccctca gcgaactggg cagcacgggg agggtttggc tacccctgcc catccctgag 1860 ccaggtacca ccattgtaag gaaacacttt cagaaattca gctggttcct ccaaaaaaaa 1920 aaaaaaaaaa aaaaaaaaaa aaaaaaaaa 1949 2 340 PRT Human 2 Met Ser Ala Asp Ala Ala Ala Gly Ala Pro Leu Pro Arg Leu Cys Cys 1 5 10 15 Leu Glu Lys Gly Pro Asn Gly Tyr Gly Phe His Leu His Gly Glu Lys 20 25 30 Gly Lys Leu Gly Gln Tyr Ile Arg Leu Val Glu Val Asn Gly Glu Asn 35 40 45 Val Glu Lys Glu Thr His Gln Gln Val Val Ser Arg Ile Arg Ala Ala 50 55 60 Leu Asn Ala Val Arg Leu Leu Val Val Asp Pro Glu Thr Asp Glu Gln 65 70 75 80 Leu Gln Lys Leu Gly Val Gln Val Arg Glu Glu Leu Leu Arg Ala Gln 85 90 95 Glu Ala Pro Gly Gln Ala Glu Pro Pro Ala Ala Ala Glu Val Gln Gly 100 105 110 Ala Gly Asn Glu Asn Glu Pro Arg Glu Ala Asp Lys Ser His Pro Glu 115 120 125 Gln Arg Glu Leu Arg Pro Arg Leu Cys Thr Met Lys Lys Gly Pro Ser 130 135 140 Gly Tyr Gly Phe Asn Leu His Ser Asp Lys Ser Lys Pro Gly Gln Phe 145 150 155 160 Ile Arg Ser Val Asp Pro Asp Ser Pro Ala Glu Ala Ser Gly Leu Arg 165 170 175 Ala Gln Asp Arg Ile Val Glu Val Asn Gly Val Cys Met Glu Gly Lys 180 185 190 Gln His Gly Asp Val Val Ser Ala Ile Arg Ala Gly Gly Asp Glu Thr 195 200 205 Lys Leu Leu Val Val Asp Arg Glu Thr Asp Glu Phe Phe Lys Lys Cys 210 215 220 Arg Val Ile Pro Ser Gln Glu His Leu Asn Gly Pro Leu Pro Val Pro 225 230 235 240 Phe Thr Asn Gly Glu Ile Gln Lys Glu Asn Ser Arg Glu Ala Leu Ala 245 250 255 Glu Ala Ala Leu Glu Ser Pro Arg Pro Ala Leu Val Arg Ser Ala Ser 260 265 270 Ser Asp Thr Ser Glu Glu Leu Asn Ser Gln Asp Ser Pro Pro Lys Gln 275 280 285 Asp Ser Thr Ala Pro Ser Ser Thr Ser Ser Ser Asp Pro Ile Leu Asp 290 295 300 Phe Asn Ile Ser Leu Ala Met Ala Lys Glu Arg Ala His Gln Lys Arg 305 310 315 320 Ser Ser Lys Arg Ala Pro Gln Met Asp Trp Ser Lys Lys Asn Glu Leu 325 330 335 Phe Ser Asn Leu 340 3 24707 DNA Human 3 gtgttgtgaa aaaaaagaga aatccctggc tcctggagct ggtgggagac aagattaagc 60 aaacctcccc tgacatgtat ccctttgacc ccaagctctg cctcctccct gaccacccat 120 gccctttcct ttaacttctc aaacagatac cagggcctaa actgctttac ctcccctcct 180 actgagtcag gttaggtggt gggaggtcac ccatttccga gttaaaccaa tgcaatatga 240 gtaaaacaaa gtcatgtggg tatgtctggg gtagagagag gggtagcaag ttcatgtgtc 300 ctccttggtc acatatctcc caaagctctg atccctgcca tgggaagtgg acaggaaaca 360 tgaggtcatg acctgcaggc atctttactg cagctctgcc ggcctggagg gggagagggg 420 gaggaagaag tatgcgctgc acatttctga ggctactgca tttgctttca aggcagaaat 480 cttgctctga gcagtcagcg gctccagttt gggcccgata aggaagttct ccgtggcctc 540 cctcaggcag agcagggagg aggctgacat tgccagtctc ttctggggcc caaggcaggt 600 tgcaggagat ccaatcccat agacagctct gggcctcttg catttgagtt tttcagaatt 660 aaactgcagt attttggaaa gcacatcctg tccactgttt ctttgaagtg agtggggggg 720 gggggtcttg ttgaaggaat tgtcattcac tgccaaaatc attccatcct ccttcctcag 780 tgtctgtcct cagatggtca gctccccgct caacagactg tctcccgcct ctgtgaccag 840 cctctctttg gcaagaggga gctagaaggc tttacagtcc taatcatttt tctgttggaa 900 aaaaaaaaaa aaaaccaagg ctcctttccc tgtggcgtgt acccagaggt tgattacctg 960 agtctgtcct gcctctcccc accccacctc cctagccaaa cgctgctgcc aaagcccacg 1020 ctattgccct agatggcctg tcttcagcgg gctgcccctc gaggtcccag gctctccgcg 1080 gagccctcac cttcccagca gggatcagaa cctgcactcc tctatgcgag tcctgggaca 1140 gcacaaagtg gattagggtt agggttccca caaacggaaa aatgttattc aaacaactct 1200 gtagggtccg aggaggccct ccgtcttaat tctcgagact gaccggccct cgctgccccg 1260 agcgggagca gttgccccgg caacagccgc tccctctcaa ctggagctgc acccaggctt 1320 tggctaaagg ctgttaaaac gttggccagg tgcggaggct cacgtctgta atcccagggc 1380 ggatcacctg aggtcaggag tttgaaacca tcctggccaa catggcgaaa tttcgtctct 1440 actaaaaata caaaaattag cggggcgtgg tggtgcgcgc ctgtaacccc agctgctcgg 1500 gaggctgagg caggggaatc gcttgaaccc gggaggcgga ggttgcagtg atccgagatc 1560 gcgccacggc agtccagcct gggcgacaga gcgagactcc gtctcaaaaa aaaaaaaaaa 1620 agttagggtc ctttacccga gggccggctt tcctcactcc ccgccacagg taggggaaac 1680 caggccggag ccggcgggcc cacccgccca gaaccgggaa ttcggcgagc cccgcccctg 1740 ccaccccagc gccggccgct cggtaacaaa cacttccact tcctgagcgc tagtcttcgc 1800 ccgccgcggg gcgccgcgcc gagcgcaggc cccgccccgc gcgttcccaa tggccggcgc 1860 cgttcacccg gccggagcgc ccaggcctgc agccccctat tggcccgcgg gaggtcccca 1920 ccctcagcgc ggccccgccc ccggggtaag gagccggggc ggactctggg acgctcagac 1980 gccgcgcggg gcggggattg gtctgtgctc ctctctcggc tcctcgcggc tcgcggcggc 2040 cgacggttcc tgggacacct gcttgcttgg cccgtccggc ggctcagggc ttctctgctg 2100 cgctcccggt tcgctggacg ggaagaaggg ctgggccgtc ccgtcccgtc cccatcggaa 2160 ccccaagtcg cgccgctgac ccgtcgcagg gcgagatgag cgcggacgca gcggccgggg 2220 cgcccctgcc ccggctctgc tgcctggaga agggtccgaa cggctacggc ttccacctgc 2280 acggggagaa gggcaagttg ggccagtaca tccggctggt ggagcccggc tcgccggccg 2340 agaaggcggg gctgctggcg ggggaccggc tggtggaggt gaacggcgaa aacgtggaga 2400 aggagaccca ccagcaggtg gtgagccgca tccgcgccgc actcaacgcc gtgcgcctgc 2460 tggtggtcga ccccgagacg gacgagcagc tgcagaagct cggcgtccag gtccgagagg 2520 agctgctgcg cgcccaggaa gcgccggggc aggccgagcc gccggccgcc gccgaggtgc 2580 agggggctgg caacgaaaat gagcctcgcg aggccgacaa gagccacccg gagcaggtaa 2640 gcggggcccg agccgcgcag gctggcatgg agtgggagga ggatccggag agacccaggt 2700 gccccggccg tccagccccg cgcccgccgt cgtttttctg aaactcgagc tgcgaggggg 2760 agaccgcttc cgcccgccga ccaggcgccc tgacacatcc taggcaggcc tggggctgcg 2820 tcccgcgacc tcctctcctt cccaggctgt gctgggagct tgagcgcctt tgccgcctgc 2880 acctcttgtt ccctggcctt tgggagggcg gcgcagggga acccagcccc cttccctcgg 2940 gtctgtgggt gtctgctccc gttcctcgga atcccccaat cctgctcctt ccctggtgcc 3000 ctctcctcgt tcaccccagt ccgcagatgg gccggggaga aagggctctc cgccccagag 3060 gtgccagctt cgcccgccac tcctacttca aaagctagag gaatagcatt actcctcctg 3120 tgtggagccc cggcgcgagg aggccctctc cgcagcccgc cggtgtgtgt cctgaacttc 3180 agtcctgctg gacttcatcc tcccggagtc ctgtgtgact tctaagggag aggaagcccc 3240 accatctcag ggcggtgtgg gggttgcctg gcagggagga ggagccaagg catctcggag 3300 tggtggttct gccacctcag aggatttacg attctcagga atgtgaagag taagcgccta 3360 gaactgtatc tgggagggaa ggggaaagac cttggaagaa gaaaaagatg gccatgggga 3420 ggagagagag ctcctggggc tcacggccct gtgtgcgcca gcggagctca cggtgagccg 3480 gtggtcttgc cctggccttg ccatggtcac tctggtgccc acacagccag caggccggtt 3540 gctttttgcc catgtttggt gtttgcccat gggatcccag tttggatcag ctgcaagagg 3600 ggcagctggc aaaataggag ttgtgctgag cctcctgtcc ccagagagtg gagtggtgca 3660 ttgtgtaggg cagggcgtga gcctcccagg accccttcag cttgcaccac cagctctccc 3720 tggtggcagt gtctgatggg agtgtccgtg gggctacgag gagcctcctg gacagagaaa 3780 gggcacagtt cggcattaag gtagaaggca aagccctaga ggatcaactg gtttggtgga 3840 agtgaggtca ccagaagccc tgtccagcca gagctgggtg ctgggaatgc cagggccacc 3900 aggctcccac ttcctgcccc tgccaagctg gcattggcaa ggggaggggg aagggatctg 3960 agcacccgga gcaaggaggt tgatgccagg gagttggggc cgatgatctc cagtcccagg 4020 cagaaatcca gtgtgaactt tgtgctgagt ccatcgatcg atttgacatt cttttttttt 4080 tttttttttg gactttggca cccacacaaa tacccaccca cccccacccc ccacaagttg 4140 cagtctcgtt cagctcctcc ctttgccctt ttggttatgc tgctgggtgc cagagggcct 4200 gggaggcgaa cgtggaggcc tggcatagca cacgtgcgca aaggcagcag ctttgagctt 4260 cctgggtgag cacagactca gtattcctgc atgttttgga gggacaccta catttgtgtg 4320 tactttgtta ggccaggaga tgagaggaga gccaccagca aatcctgggg acaccatcac 4380 cagccaagtc ccaggacttc tgagttctgg ttctcccatc tgtgagatgg aggcattggg 4440 cgatgtgatg cctgccttct ctagaattat agaaatgacg tgaagcactt gagcccctta 4500 ctaaatgctg aactgcactc cttcatgcct ggcgtttgaa tcccagctcc tctaaactga 4560 ctcctagtca acaccattga gtccttctag cagaaaatcc tctcctctca atcctgtgtg 4620 ttgagcatac agacacccat gtggttatta gaaaaaatgt taacagacat tgtaagagtt 4680 cgtttttcta aaacacaccc gtttcctaca taaagcatca aatggaggtt tgccaattcg 4740 ttttagtctg aatttgtgtg caaggtgggg ctgttccctt gtcttcatta gccctccact 4800 tactaagtga tgatatcagt ggaaaagtgt ggcgcagaga gtggttggct gggtggcctc 4860 tggctgggaa gaccagctgt gtccagaacc actcagggca gaggctgagg ggtgcctgta 4920 acactggcca tccccgctgg ggagtctagg cctaaggagg tggaagtggc tctccagctc 4980 tgcccaccgg ctttgctttg tggatggcct tccccgcctg ccccgtgggg agagaggagc 5040 agcaagaccc gccctgctgt tcccctgctt aaagccctcc tccccccatt caccaccagt 5100 cacaggatga ggcctaaacc cttgagtctg ggttcagagt gccggccggg cagagccgag 5160 ccagctcagc tgtactagcc aggctgtgcg aagccaagtt acctcacctc tgtaagcctc 5220 catttcctct tgtgtaagtt gggggttatg gcagctacct cggaattgca tgaggctgtg 5280 tgtaaagcac gtagctcagt gcctggcact gagtccaggc tcagccagcc tttgcagttg 5340 gtattggaat gaatacatat ttcatagtga tcattgcaca cctatcatgg ggcagctagc 5400 gctgggtcag cctgcctagt tgggcaaatg ccactgtggc caagcctggc acacagtcag 5460 tgcctgatcg atggtcactg tgggttaaga atgatacgtt gtggccaggt gtggtggctc 5520 acacctgtaa tcccagcact ttgggaggcc aaagtgggag gatcgcctga gcccaggagt 5580 ttgagaccag cctaggcaac atgacaagac cctgtctcta ttaaattttt tctttttttt 5640 ttttaagagt tatatgctgt tccatggccc ttctccatct ggcagcctgt tctcaccact 5700 gcctccctac ccccaaaacc acacccaggg tccgaattcc cctctaacct ctaagccttc 5760 acaccctgac acttctgtcc cctcccaggg agcctgtcct taatccagta ggcagagtta 5820 gcatttcctt tccttcactt tttttgtttt tgtttttgtt tttgtttttt ttgagacaga 5880 ttccacccag gctggagtgg tgtcgcaacc tcgcctcgct gcagcctcga cctccagggc 5940 tcaatcgatc ctcccacctc attctcccta gtagctggga ctacaggcat gcaccactat 6000 tcccagctaa tttttaaatt ctttgtagag atggagtttc accgtgttgc ccaggctggt 6060 cttgaactcc tgggctgagg tgatctgcca gcctcggcct ccgcctcctc tttgtcctta 6120 catcttttat cgctctaaac acacaatatt ttaatcatct gtttgtgacc tttttctcca 6180 ctacgtggta agcccaaggg catcaactgt ggcctatttg tgtttctatc cccagcgctg 6240 tccttggcac atggtaggga ggctctcaaa aacagttttt gaattaatga atgaatatat 6300 aacaacagtc agggaccttt gtcctcattc agctcatccc gccccgccca gagattggaa 6360 ttccagatag aacctaccca ctcttctttc ttgcctgcta gcagcttcta tttgaaaact 6420 agaaagccaa aagtccaggc tttggggtca gccagatcag tgtttgaatc ccagctctgc 6480 cgtgtgctgg ttctgggatc ctgggagttc atttttccct gcaaccctca gcctccacct 6540 ctgtgaaagg cactgcctgc agggttattg ggaagatttg gtggcatgac aggtttgtaa 6600 atgaaagtgc tagtttgggg tgtttcctgc cagccctgac cctgatccca gagtcagagt 6660 gcaggcaccc cagggagcaa ggtgggtggt ggggcacagt tagctggagg gccatcaacc 6720 tctttcccag ggcgggcggg ccattttaca ccaaccaagt tggagctctg gtagggcaga 6780 acagagcttg agctgctggg tgtttggatt tgaaatggac agggtatgtg attgtttgtg 6840 tgttgggatc tctttctttg ctggcaaaac agtgtagccc ctggttctta ccttcaagct 6900 cctgttaact cagtaattct ggagatgatt ctcttggaga tagatggggg cttcctggcc 6960 gggcgcagtg acttgtgcct gtaatcccat cacttcggga ggctgaggcg ggaggatcgc 7020 ttaagcccag gagtttgaga ccagcctggg caacatagtg agacctcgtc tctaccaaaa 7080 aaacaggggg aggggggtga atgggggctt ccttctcaag gaactccata tgtctagatg 7140 gggtccttct cccctttccc agatgtccca tctcaatggt ccctgggaaa gtggggtggg 7200 aaattaataa gaaactcagg ccaggcacgg tggctcacgc ctgtaatccc agcactttgg 7260 gaggccgagg cgggtggatc agttgaggtc aggagttgag gtcaggagtt caagaccagc 7320 ctggccaaca tggtaaaaac ccatctctac taaaaataca aaaattagcg aggcgtggtg 7380 gcaggtgcct gtaatcccag ctactcggga gtctgaggca ggagaatggc ttgaacctgg 7440 gaggtggagg tttcagtgag ccgagatcgc gccactgcac tccagcccga gcagcagagt 7500 gagactcatc tccaaaataa aaaaaaaaga aagaagaagc tcagccgagg tcttgttggt 7560 gctgcaggac tctttacagg gagaaaccag agctctagac cccaataaca aatgttcctc 7620 tgttttcgtt gctctaagat agagtggccc tacctggaag gaaacaggtt tgtacagtgc 7680 ctttaggcac tctacctatg acataaagat gttgacatgc atttctttct ttctttcttt 7740 ctttctttct tttttttttt tttttttttt tcctgagaca gtcttgctct gttgcccagg 7800 ctggagtgca gtggtgtgat ctcggctcac tgcaacctcc gtctctcaag ttcaagcaat 7860 tgtcctgcct cagccttcca tgtagctagg attacaaaca cccgccacca caccctgcta 7920 atttttgtat ttttagtaga gacggagttt caccacggtg gccaggctgg gtggcacctg 7980 tcacaacacc ctgctaattt ttgtattttt agtagagatg gggtttcacc acattggcca 8040 ggctggtttc aaactcctga cctcaggtaa tccacctgcc tcggtctccc aaagtgctgg 8100 gattataggc atgagccacc acacccagcc aatatgcatt tcattgtccc ccagacaaat 8160 tagctattgt tttctctatt ttgtaggttg acagtaagtg ttcaatacat gatagctact 8220 atttgtgtct agcactgtgc taagtactaa ccttgcctgt ctcatttaat ccacatgacc 8280 accagtgagg cagatattgg ccccattctc tgcctatgga ggctgaggct tgatcagtta 8340 aggtggccat ggcgggtccc aggactctga gctagcgact cctcccacac agcgcagggg 8400 tgtgtcttgg aacccaaccc tctgactcta gtgcctgcac agccatttgg ccacatgagt 8460 aggctgtgct tgaagcctga ctctgccagg tgtttctgcc catcatattc tgagccacag 8520 ctggaccatc acagtggtat aaattcaggc tgcttgaact ggcttcctgt aaggatccag 8580 ggcttccagg tgctcctgtc cctagcaggg catccttgca gtgcctcagc ccataagtgg 8640 caggctcctg tccccatccc ttctcacgtg atgtgatagg tcaatctgga atctggtaac 8700 caagtatcta cccccgccct cactccccag aacctgcaca gagagccccc ctcaagggtg 8760 gtcttcactg ttgaccgaag ccttcccctc atggtaggac aactcaccat gtccccagac 8820 tcctaggcaa tgtttaggtc cctaggccct agaaaaggac aagagttaca gattccctga 8880 ggtccaggtc acctcggggt ggtgtgggaa cacacagaga atgctggcca ggttcaagtc 8940 ccactcactg ctgggtgctg agagctctga attctccgag acaggctgca gcctctccct 9000 gtgctaaggt caggggaaca gcaatggcca ggggtattta gaggagtgtg tttggggcag 9060 tctcaggcag cacaggtctg tgctgtccgg gcagcagcca ccagcttcgc gaggctcccg 9120 ggcactcgag atgtgctggt ccacatgcag acgtgctgtg tgtgcaaaat gcacaccaga 9180 ttttgaagcc attgtacaaa taaaaggatg tacaatagct caattgttaa aaatggatta 9240 cacattgaaa tgataatatt ttgggtacat tatgttaaat aaaatattca attctactat 9300 ttttcctgtt tttttttttt ttggaggcag agtctcgctc tgttgcccag gctggagtgc 9360 agtggtgcga tctcggctca ctgcaagctc cacctcccag gttcaagcga ttctcctgcc 9420 tcagcctccc aagtagctgt gcaccaccac gcccagctga tttttgtatt tttctttttt 9480 agtagagata gggtttcacc atgttgacca ggctggtctc aaactcctgt gctcaagtga 9540 tccacttgcc tcgtcctccc aaaatgctgg gattacaggc atgagccacc acacctggcc 9600 tatttttctt ttctttcttt ctttcttttt tttttttttt ttttttttga gacggagcct 9660 tgctctgtca cccaggctgg agtgcagtgg cgtgatctcg gctcactgca acctccgcct 9720 cctgggttca agcggttctc gtgcctcagc ctctggagta gctgggacca caggtgtgca 9780 ccaccacata tgtctaattt tcatattttt agttgagaca gggtttcacc atgttggcca 9840 tgctggtctt gaactcccga cctcaggtga tccacctgcc ttggcctccc aaaatgctgg 9900 gattacaagt gtgagccact gcacccggcc tatttttcct ttttcaaatg tgatgactag 9960 aaaatcttac attccacaca tggctggcat tatatatcta ctggacagtg ctgctctgga 10020 tctgagctca aatcctgcct ttgcccgtca gccatgtgta cttgagcaac tgactccagc 10080 tttttgaagc tgggtttcct tttctgtgaa atgctggcaa taatactttc cttttaggac 10140 tataattggc tcttaagaag aaaacacatg taaagagctc ggtgcctaac acatagtagg 10200 tgttcagtaa atagcagctg ctatttgtta cttcagtgtt gagccccaat ttcacaagct 10260 ccttgaaggt tcttaatagc cccttgggac aacccctggg ggttaaatct agagacttct 10320 gggaggaggg tgagggatga ggaaggatgc agcagggaag gcatggggcg gccagccctg 10380 gatggtgtcc agccgggctg cagcccaggt accagcctgc tccatcccag gcttgcccag 10440 ccctgcagag acctgagcca gactctgccc cctcttgccc ccgtgggtct ccatggctgg 10500 cacagggaga agaggttact agccagaagg agaacctgtt tcagacctct gagtcctggg 10560 ggtgtctgtc agaccagcag acctcacttt agtttgtgtc tccctcctgg ttcccaccct 10620 accagactcc agggaaagct cttcccactg agaaatagga aggaagagat caagggctag 10680 gagcagtgag tgactttttt taaaatggac acatccaaca ttcccctgcc gcccgcacag 10740 acctggaggg atgtggaggg tggaaggggg tcccatagag taaaatgagg tgacatcagg 10800 gccactacgc agggggccag tgacagccaa gccccctgta cctcacctag ggtaaggaga 10860 agacaccttc tgcctagagg tgtggacact ggctgagtgc ctggagcccc tggtcacttc 10920 ccagtgcccc caggctgccc gtcacacagc actccaggca gggccagctg tctccatcct 10980 ggcttccttg aagagctggc accaggccca gccctgtctc cagaatctgg ggaacaaact 11040 gagctgtctc ccagagcaaa tatggctctt acacccaaca tactcctgac acacacaccc 11100 cagcccctga gaacagccag ggttccttcc tgcccccgcc gtcctgcccc cttccatcct 11160 cttgttgggg ctgattttcc tgtattgggc atggggcact ggtcttcttt gcataaacaa 11220 gagccctggg ctgggggagg ggcagtcttg agaggtgggg ttacctctgt tgttcccctg 11280 gacacccaag tggtcttcct actcctccag cccctttcct ctggggtcct gagctagctg 11340 gctcgaagga agcgggctgg gctgtttctg tagggcccat caccatggca tcagccaggg 11400 aggggctggg cccaatgggg cgttggcccc caggctccca cagccttccc tgaagggagg 11460 aaggaggagc agggagcggc cctgggcctg ttccccagag acggtcactg ggattagggt 11520 atcgactttc cccagctttc caggtgactg ctggtggcgc ctattgaatt ccgtgcccgc 11580 catccgctcc ctggactgcg tgggtggagg tgatggggcg gccacttagg gcgcggtgaa 11640 gctccctggg ggaaaggaca gatgaaggga ggaagcctga gggtggagga ggccccggga 11700 gaagggtgtg ggggcagctc tccgggcagg gtcttttctg gaaagccagc cagcattttg 11760 tgaaagtgag agtctccagc ctgtccagcc ctgggtcacc ccggcccagc tgcgctgccc 11820 atcggctgca tggcctcatg ttccctcctg ttgattctgg ctctctgcca gtctcctctc 11880 tgagcagatc ctgattttgt cttgaacttg ggtctcccag gctgtcgtcg tcactggagg 11940 tcccactttg ccctccggaa ctcactgggg ctatgttcct tttgagtctc ctcctcccac 12000 catgcttcgg acccctctgt agctggctcc tcctaagatc ctgggggcca cacaccaagc 12060 ccaacatgcc tggacgtcac tcccatttat cccctttccc cagaggcaga acccagccct 12120 gccccagctt ggcggtgagg gtccaagagc aatggtggct gagctcggcc aggtacccca 12180 cacctcccca tcttctcctc tcagtgctgg gcctggagac cacaggccag gccccacaga 12240 tccccagggg ggctgcccca agggactccc ctgccagccc cagcctccac ccaggctggg 12300 atgggctggc ttgatgggct gactcagcaa ctggcactga cacctcctgg agacagctgg 12360 gaggctcctg ctagggaggg gtgaggagga acaaggctgg tctcccttga agagtgggaa 12420 ccccccttcc catgtgaggg gaggaagtct ctagggactt gggaaggagg ctctgcaggt 12480 gcatcggagc tgtctaaatg ctggtggggt ctaccggggg cttcccatct ccttgtccag 12540 ccaccccctc aggtcctgga gccgccacag agccacatct agacagaaca gtggagcagg 12600 tcccagtgca ggggaaggaa acagagggca cctgagcccc tcaggctggg taccctgttt 12660 ccccttgaac tgcacgactt ggtgctggca gagcggagca aggctcaaag caggccctgg 12720 agctttggca ccatatgtca ctaggcgagg ggcactgggc tgaagatcag taagggaagc 12780 ccacagcaca gagaccaggg ccagaggatg ggcatcccaa aggcagctag cacagccctg 12840 ccctgccagc cggggggggc gcccagtagc cacctgcccc cagtccagcc atgcccctgc 12900 cctgtcaccc acaccaggct tcatccccca gctcaggctg cagctccagc tgcgagaggc 12960 cctgagcgcc cggaaggaag gctgtggtag gttgtggttt cttcagctcc cagcctacct 13020 ccagcagagg ctgcagccga tgccctggag agagcccctg tctgccctct gctgcctgac 13080 tcctgttggg tcctcacctc ccaatgggtt gccccattct ggacttggat ccttaaggag 13140 caccctgcag tgtggtggaa ggaggacagg ttccaagtca gagaccaggg ttggagttct 13200 ggctgtgtga tgtcaggcag tgggctccac atctctgagc ctcagtttcc ccatctcaaa 13260 aacagctgac aagccccagt caggttgtta tgggaaggtt tttgggagcg cagggacggt 13320 ggagggaggt tggaggaggt cactagagct gccccctgcc tgtcctgaga tttcaccctc 13380 cccactttct cagggtgtct ggctgaagca gaaagctgcg cctgtcctgg tgacggcgac 13440 tggtcagact tgtttctgct cacctaggcg gcccggggga gggcctggct gggagctttg 13500 tggagggatt agctctaggg gagaagagcc tcacttactc ccggaacaag atcccacagg 13560 gctgtgggag tgccgggggg tgagcctggg gcaggtgaca gccgaactag ctgggagtgg 13620 gccctgcagt gaggcagggg gtgggccagg gagaacaagg caagaggagc ttcattcagg 13680 gttcctgagc ctttgtgagc cactcacgtt tttaccactc acttaaccgt ctttgttgtt 13740 ggggtgaggg gtcctcgagc ctggatttgg gtatgaaaac ccaggcaaga aagacctgcc 13800 caagccttta aaggaatgca aagtcatcct ctagccaccc ccagagatcg aaaggctggg 13860 gattgagtct cctgcagatg gtggcggcct cctggggctg gcaagttggg acagaggccc 13920 ataagccctc ctgggcgcgc cttcccaccc ctctcggccc tctccactcc cagctgggga 13980 tttgggtttc agagcagcct ggcacacaca cccccacccc accagaatct cactcccagc 14040 ttcctatgac tattcattag tattcacaac aatgggaaag tctgggtgtg cacagggatt 14100 ttttacagtt agaaagtgtt taagtcaatg acctcactgg gcctcagcaa ccctgggagg 14160 cagatggcag tcagaatgat ccataaatga cctgccccag gtcacacagc tcctaaacag 14220 gggagctgga acctggctgg gagccttgac tatccactgc tcattgtctg atgtgctgag 14280 tgataacaca gggccagcag gctggacagc agccagtggc cctgcaccag gcctggagga 14340 gggggagagg gacagggact caggcgtagt ccttgggcac aggcttcggc tgcctacacc 14400 catattccct ccatcatact gtgtgtccag gcctgcagct tctaagctga gctgcttact 14460 ttggaccaag catgttggaa actgttttca gctgagtcca agcactcgaa atctgcgtgt 14520 gcccttttag taggtcacac cctccaggcc acagccacac tgggctccct ttagcccagc 14580 cctgcttttc ccagtcccct ccccattcag gctcctctgt ttctggggtc tctgtgcttc 14640 cgttgccacc tctgcccttg ggcagcggtg tcgggggagg aggtcctgga acgcctgagg 14700 atggccccgt cttgtccagg tccccttgct gtgctgaaca ataagttttt ggttatctgc 14760 tccttctggc tcccttttgg ctgggccatt cggcttggtg ggggtgagag agagttcgag 14820 tatatggagg aaggctttac tgtaaacaca ctgaccagct ttcagtaaac acctgcctct 14880 tcctcccctg ctgcagagaa cggggtacaa aggggctggg ggtcctcagt catatgcccc 14940 aggtccccag ccaggctgac gggtgccaca ctctgccccc tctccaggag ggccagactg 15000 agccaccaat ggggagggat agaggcccga tcaacacagc atgtgattca ccttagactg 15060 tcagaaggga caggcatgtg agagctaacc tggtcctacc cctcatttgg cagaagagga 15120 aactgaggct tggacagagc agtgttttat ccacatcacc taagcagtta gctgcggtgc 15180 tgggtatcca cacaagtctt ttgagtcctg ggccagaacc ctctcaaatt gctgtgtagg 15240 gatctaggca aatttgcatc ctcccaaacc ttccttggtt ggggagatgg ggacaggcag 15300 aggacaggga aggcagaacc tggggcactg tggaatagcc atggaccctc ccctgtccct 15360 gcagcgcgag cttcggcctc ggctctgtac catgaagaag ggccccagtg gctatggctt 15420 caacctgcac agcgacaagt ccaagccagg ccagttcatc cggtcagtgg acccagactc 15480 cccggctgag gcttcagggc tccgggccca ggatcgcatt gtggaggtga tgcttctcgc 15540 tctcttccta tctgactgcc cccaccccct gcagatcagc agcacctggg gcagccatca 15600 taccatcatg ggcttgatta gcccacgggc atagccaacc tggagctgct ggatggatgg 15660 gtggatggga gggagggagg gaagggtggt ggatggatag acggaaggac agatggactg 15720 actgatggac tcttctctta cctccactcc cctgggactc ctccttgcca agcagataca 15780 gtgggcagcc tgtgattcac ccatcgatca ggggagatgt ctggggccat ctccaggagc 15840 ttccctgctg ccaggcacac agagtggggt ggcagtaaca aaacccctct cctgagttag 15900 gcctttattt atttagtttt tttttttttt aaatggagtc tcgctttgtt gcccaggctg 15960 ggggagtgca gtggcacaat cccagctcac tgcaacctcc gcctcctggg ttcaagcgat 16020 tctcctgcct cagcctcctg agtagctggg attacaggtg cccaccacca cgcctggcta 16080 atgtttgcat ttttagtaga gacaggattt caccatgttg gccaggctgg tctcaaactc 16140 ctgacctcaa gtgatctgcc cgcctcggcc tcccaaagtg ctgggattcc aggcgtgagc 16200 cacctcaccc ggccccgagt taggctttta gactcgtgca ttcagggctc tggtgctgtg 16260 ctcctgggag aggaaacgtg aaccaaatgc tcaggcaggt cccagaatca cagtcttaga 16320 accttagagc tagagatcag ctctcccaag acttctcatt ttacacgtga gaaaaccaag 16380 gcccagagaa ggaagtcgga cagcaagtcc ttacccaggc tgggccccac cccggcctcc 16440 cagctcgctc ccatcccatc tctcccagct gtgtttgttt agtctcgtct ggatattttc 16500 gttggccctt cccatgtatc ctgcctcccc agctgactgt caatgggagg acccccagcc 16560 tagttcagga ctctgcagaa atggcggggg tcagtatccc caggttgtga actgcaaact 16620 ggctgagaac caaggtggtc acccctgccc ccaacccccc tccacttacc tgccccagaa 16680 ccaaggcctg tgtccgtaac gcctccccga ccctgccctg caggtgaacg gggtctgcat 16740 ggaggggaag cagcatgggg acgtggtgtc cgccatcagg gctggcgggg acgagaccaa 16800 gctgctggtg gtggacaggg aaactgacga gttcttcaag aaatgcagag tgatcccatc 16860 tcaggagcac ctgaatggta agccaggtgg ggccactggc cgtcctgggg ctggagcccc 16920 ccaagtcagg gatgtgagcc agggctaaga ctgctgggtc ccaggcgagg ggtgggcagc 16980 ttcccggcat gggtgctccc tcctcctcct tcatgggagg cccagaggtg tgggctgggg 17040 gagcgggggc ctagtgtagg gagtggcagt gggtttctga ggacggcttg tgatggggtc 17100 agctggcatg aggtcggtga gagagatgga cagatcttct tattcccggc caaagctgca 17160 gccccaaagg aggcccaagc ccccagtcct gtccccacca gcagactttc agggcagtgt 17220 caatgtgagg aaagggttaa ctccggggag gcctccagcc tttgcttggt cagatcagag 17280 tccagtgacg ggggccggtg cctccccctc cctcctctca ggttgtcttg gaaactcagc 17340 cttgctggct ctagagagta gtgggtcccc cttcaatccc tgggcccctg ccctccccat 17400 cccccaccat caccctgcca agcctggcgc ctccccctgc ccaggcccga gccaaacagg 17460 gcagggctgc tgcaccgggg cagggagggg ttaagcatgc tctgctcctt ggtctggact 17520 ttctccctgg gaagatggct ccctggagcg ggcaggggtg ctcactgggc catactgagg 17580 gtggaacctc ttgggacttt gcagtgggtg gggcctcctg gagactcaag gttgtatgtg 17640 taaaaggaga ttcacacaaa ggtcctgcga ctcccatcgc cagctgcaag ggcccactgg 17700 ggaggccctg tccccttctg gcgcaatcca aggcctgagg ctcctagaag aggggatggc 17760 ccctggaagt ccctagctgg ctctgggcat gggaggtggg gtaccgcctt acccttatct 17820 cccaagttca tggaaaccag gtaactcaga taatcccctt ctccactgag ggggagacta 17880 aggcccagag aagccccagc cttcctccta gggatctgat acgaggaaga cacagctgga 17940 atctggattc cactcagctc ctgggatact gtcccctgct tcccccaccc cttcaccctg 18000 caggtaattg gccccctcag acattcctcc ctcttctgcc tgtctctcgg gctagagggg 18060 ctgcagcctc tggccaaagg acctgaagag ggagaggcct ggggacagtc ccctccccgc 18120 agtccctcca ggatggcatc ctcacccttt ccatagggag gaatgccccc ctctccggct 18180 gccagggttt caccctgacc actgtgagct gatggggagg ggacagtgag tgaccctgtg 18240 tcccagcagc ctgtccttgc ccggggaagg ctgtgggtgt caaaagggag gagaagatga 18300 gagtcaggta tctctggctg tgtcctggac tggggacagg gaaaagataa ccaggaattt 18360 taagctaaga gttcagaaga agccctacac tgaccagtcc ctggagatgg aacagccacc 18420 cctgaagcca tgtgggaccc ctaccctgag tgagcccagt gaaggtgacc ccagacctgt 18480 cttctctccc tctgacccct ccaggtttcc cactggctgg gggaggggat acccaggaca 18540 caccccttga gccttctcgt cccccctcat ttcctgattg gcaaatagga gttgggatta 18600 ttttctcttt ttttcttttt ctttttcttt tttttgagat ggagtcctgc tctgtcaccc 18660 aggctggagt gcagtggtgc gatctcggct cacagcaaca tctgccccta gattcaagtg 18720 attcttctgc ctcagtctcc tgagtagccg ggactacagc catgcgccac cacgcccagc 18780 aaatttttat atttttagta gagatggggt ttcactatgt tggccaggat ggtctcgatc 18840 tcttgacctt gtgatccgcc cacctcggcc tcccaaagta ctgcgattac aagtgtgagc 18900 caccgcgcct ggcctttttt tttttttttc cggacacagt ctctgtcacc caggctagag 18960 tgcaatggca caatcttggc tcactgcaac ctccacctcc tgggttcaag caattctcct 19020 gcctcagcct cctgagtagc tgggattaca ggcgcctgcc accatgcccg gctaattttt 19080 gtatttttag tagggatagg gtttcgccat gttggccagg ctggtcttga actcctgacc 19140 tcaggtgatc cgcctgccac agtctcccaa agtgctgggg tcccagtcat gagccaccga 19200 gcccagacta ttttctcttt cagttgaggc aggtactaac tatagtctgg ggtgtggttc 19260 caattttggt gtcatcatca tcacctggga agcgtttgga aaatgttgat tcttaggcct 19320 gcccactaaa ccagaatctg tgctgccaag gtccaggaat ctgtacttta acaagcttcc 19380 cacgtaggag ttcccaggta ggaattccca ggtaggaatt aggcagccat cctcgaattt 19440 catccccatc tgagacaata cattttttaa acaccttgtg acccatcagt gcctcataag 19500 atcaatttag tagatagaga ccagcaggtt ttttggtttg ttttttttgt tttgttttgt 19560 tttgttttgt tttgccagag tcttgctgtg ttgcccaggc tggagtgtag tggcgtgatc 19620 tcggctcact gcaacctcca ccttctgggt tcaaacaatt ttcccacctc agcctcccaa 19680 gtagctagga ttacaggcgt gtgccaccat gcccagctaa ttttcttttt gtctttttta 19740 gtagagacgg ggtttcacca tgttggccag gctggtctca aactcctaac ctcaagtgat 19800 ccaggtgccc tcggcctccc aaagtgctgg gattactggt gtgagccact atgcctggcc 19860 cagacatttt tttgtttttt ttaatgaatg aaattaaaaa tatcaaagaa cactgtagta 19920 agggtgttgc tttgtgaaac ttttatcaca caaaatcttt gtgtgtgctg agtcatcaaa 19980 atggcaaacg taggccagac atttgctcac acctgtaatc ccaacacttt gggaggccaa 20040 ggtggaagga ttgcttgggg ctaggagttt gagaccatcc tgcatgacat ggcaaggccc 20100 catctctata aaaaaaaaat tttttttaaa taaataaggc aaatgtattc atggtgggtg 20160 gtagtcaaaa aagtttgaga tgttgaccca gggaacaaga tctaataatc tgcccaaacc 20220 caactcctac ctcctctcca cgctctattc catccccgtt ctggactcac ctctgctctg 20280 tcctttgcct aggtcccctg cctgtgccct tcaccaatgg ggagatacag aaggtaaggg 20340 cgggtcccct gtctctttgg atttcaatcc ttggggtgca tgagacagat cagaaggtgc 20400 tgtggttggt aggatagcca tctgactaag gccaaaaccc cagggtcccc agttccagct 20460 ccttcttcag ctccaagcta tagaactgcc agaaccctct tggcctctgc ctggggccaa 20520 agctgtcagg agggagcggg ctggctgggc ccctctgcag cccgccatcc tgggtgacaa 20580 gcccccgcct ccaactcccc accatacaga aggggccatt cttcccccca agaacagagc 20640 ttcagtgtct ggccacgggc agccgggccc ctgctacttc tagtctgggt tggaagaaca 20700 aagcccctcc tccccccttc cctgggcaga agggaaaggg ggtgaggccc ggggaagaga 20760 tggtggggct gggcgccagc tgaagccatg gaggaaccca ggaaggggct gggtgccaag 20820 ctgacgcccc agccaaagcc ttcgctctgg gacaggccga ctcagtcagg ccacagagca 20880 ggaactctgc agggtctgct taaaggttcc ccttccctag gagctcctgc cagggtactt 20940 ggcaaggggg agggcccttg gacccagccc tcaaccttct ttcacctcat ctttggatct 21000 agaactttcc atccaccttc ttctttcaag gccctccttt gctgcttcag ggttagggtt 21060 tcaggattcc tggggtcaaa tcctggcaaa cagtcaggtg gaccacatga gattcaggga 21120 gcaccacatg ggctccccat gcccccatgt gtccccacat tccaagaagc ccctccctgg 21180 tcactggccc aaaaggcaag ccagtaccta aaagccacat tggagtggcc cagggagagg 21240 gagcggttca gctaatccca tgatggtctg ttcccatccc atccctccag cctgagcagt 21300 agaaccagcc tgccctctga tccccaaagg aatgtaaaaa ggagccccag caggctcagg 21360 aggtgggaac caggggtggg tggccagggc atcagtgcta cctcttctca gtctgaggcc 21420 cctacttccc caggagctcc ctccctcctc aggaccccct caccccatcc tctgacaacc 21480 cacaaccctc tcctctctgc caggagaaca gtcgtgaagc cctggcagag gcagccttgg 21540 agagccccag gccagccctg gtgagatccg cctccagtga caccagcgag gaggtaggcc 21600 agccatgcgg ggggtggcaa ctgggttaca ggaagccgat tcccaggccc cacttgttcc 21660 tggcacacca gcctgccttt gaggtcacat gctgagccgc attctgttct tgtgacctgg 21720 cttccctggg cacggcccca acggagccac ctcaccaagg ctgaggacca gggagcctaa 21780 tgagggactg actcccaact tcctgccccc acttctcttt acagctgaat tcccaagaca 21840 gccccccaaa acaggactcc acagcgccct cgtctacctc ctcctccgac cccatcctag 21900 acttcaacat ctccctggcc atggccaaag agagggccca ccagaaacgc agcagcaaac 21960 gggccccgca gatggactgg agcaagaaaa acgaactctt cagcaacctc tgagcgccct 22020 gctgccaccc agtgactggc agggccgagc cagcattcca ccccaccttt ttccttctcc 22080 ccaattactc ccctgaatca atgtacaaat cagcacccac atcccctttc ttgacaaatg 22140 atttttctag agaactatgt tcttccctga ctttagggaa ggtgaatgtg ttcccgtcct 22200 cccgcagtca gaaaggagac tctgcctccc tcctcctcac tgagtgcctc atcctaccgg 22260 gtgtcccttt gccaccctgc ctgggacatc gctggaacct gcaccatgcc aggatcatgg 22320 gaccaggcga gagggcaccc tcccttcctc ccccatgtga taaatgggtc cagggctgat 22380 caaagaactc tgactgcaga actgccgctc tcagtggaca gggcatctgt taccctgaga 22440 cctgtggcag acacgtcttg ttttcatttg atttttgtta agagtgcagt attgcagagt 22500 ctagaggaat ttttgtttcc ttgattaaca tgattttcct ggttgttaca tccagggcat 22560 ggcagtggcc tcagccttaa acttttgttc ctactcccac cctcagcgaa ctgggcagca 22620 cggggagggt ttggctaccc ctgcccatcc ctgagccagg taccaccatt gtaaggaaac 22680 actttcagaa attcagctgg ttcctccaaa cccttcagcc tccgtgtgtt ccttggaagt 22740 tttgtcctct ggccttggac cccttatagg tagaaattga gaaatggtaa gccaaggtgg 22800 tctttggctg ggagggtggg gtacactgga gggagggcca tcaagggctc cctgtgaccc 22860 caagcctggg tagctttagc tagagggcct agctgcagtc ctgtaggaag gaagatgcat 22920 gcacccagcc gggtattcag cttggtgtgg tcagtgtgcc tgtgtgctgg gctgcaagca 22980 ccgattgtgg gctggggacc ccttgtctaa cggggatatt tacaagggga agtgggagct 23040 cagaccaacg ttctcagagg actctgggag gttcctttaa ttccagaagc gtggaaagtg 23100 tgtcccagga tggagctggg tttggaatgt gaggacttgg ctttactctt tctgcctata 23160 gccagtgggg tgcagaattc ccaggggcag gctgggctgg tgccagatcc tctatcttat 23220 ctggccattg tgacctgatt ggagggcaag tgtccagtgc caggggaaaa caaaccggtc 23280 cactgagccc ggggacatgc tgtgtggcta gctggggtgg aagggactgt tcaagggcaa 23340 ggtgccctgc ccccaacaaa agacccaggt ccctgatacc tttggcacct gaagcctgat 23400 ctggaggtcc aggaaggtgg cttgcaagag ctaggctgca gagccgggag gttccgggca 23460 ggtgctcagg aggaaggtgc ccccaggcta gtctccaccc aacccaaagg gacacctctc 23520 agaaaatagg ctcctgggct gaggggaatg agtaaagcag gtagatctgg aatcagggag 23580 tggacagtgt cccccaggcc agtcactatc tggccatctc ctgctccttg tcagaggaac 23640 catccaaaac ccttaccaca gcagtgccct gctcagctga cctgccccca ccccaccctt 23700 gcttctaccc tttggcccct gacagattcc cctccacagg ggtcgtggag ggcccctccc 23760 tatctcacct gctgggtggg gaagccctgg gggacctttg actcttcatt aacctgaaga 23820 ctggtggaag gaatgggtgg ccccaaggga actctcattg ctgaggtaca catggtacga 23880 agggagccac ggcaagcaac gcgttcgttt tataatgttt gttttatact gaggcatgtt 23940 ttcggttcca gttgttcaca tgcagtctgc tgtgagactc caagatcaag ggtgctggga 24000 ggccttaggc aagtcacctt acttatctaa gactgtttcc ccacctggaa gatgccctac 24060 aagcctcctg tggctgtgtt tagaaagcat gcccggcctt tcttgacagc cagccacccc 24120 agatgatggc agggcaagga agactgttag gagtcagagt gctcccctca ggtggaagga 24180 aactgggcca actctacttt gtaagccata gggtgccagg tagcccggcc accctgagcc 24240 tgtgcctcca ctgcccccgc gtggccagtc aggtgcagct gctcccagag atggagggtg 24300 aggaacagac gtgggagcac cagagggaca gagctgatgg cctgacgctc tcttcaggag 24360 ggcaccccca aggggcctct gcttcctcag tgccccctga gctttatcag cagaggggtg 24420 ttttccagcc acaaggagct gtatctaaca ctaatgcctt taaactcaag actggctcca 24480 ggagagagga ggacggacac taggttgagg ggccaggcca cactcactct ggaccacctg 24540 ttgttcccgg gtcaagttcc cagggtcaca ccagcctgcc tctgcaggac aagaggacca 24600 agctgccctt gagtggacac tgtgaggctg gggcttgtgg tagctcttca cacggaccaa 24660 acgggaaaat caggaaagct ggtagtgcct ggagcttcac tcccagc 24707 4 358 PRT Human 4 Met Ser Ala Asp Ala Ala Ala Gly Ala Pro Leu Pro Arg Leu Cys Cys 1 5 10 15 Leu Glu Lys Gly Pro Asn Gly Tyr Gly Phe His Leu His Gly Glu Lys 20 25 30 Gly Lys Leu Gly Gln Tyr Ile Arg Leu Val Glu Pro Gly Ser Pro Ala 35 40 45 Glu Lys Ala Gly Leu Leu Ala Gly Asp Arg Leu Val Glu Val Asn Gly 50 55 60 Glu Asn Val Glu Lys Glu Thr His Gln Gln Val Val Ser Arg Ile Arg 65 70 75 80 Ala Ala Leu Asn Ala Val Arg Leu Leu Val Val Asp Pro Glu Thr Asp 85 90 95 Glu Gln Leu Gln Lys Leu Gly Val Gln Val Arg Glu Glu Leu Leu Arg 100 105 110 Ala Gln Glu Ala Pro Gly Gln Ala Glu Pro Pro Ala Ala Ala Glu Val 115 120 125 Gln Gly Ala Gly Asn Glu Asn Glu Pro Arg Glu Ala Asp Lys Ser His 130 135 140 Pro Glu Gln Arg Glu Leu Arg Pro Arg Leu Cys Thr Met Lys Lys Gly 145 150 155 160 Pro Ser Gly Tyr Gly Phe Asn Leu His Ser Asp Lys Ser Lys Pro Gly 165 170 175 Gln Phe Ile Arg Ser Val Asp Pro Asp Ser Pro Ala Glu Ala Ser Gly 180 185 190 Leu Arg Ala Gln Asp Arg Ile Val Glu Val Asn Gly Val Cys Met Glu 195 200 205 Gly Lys Gln His Gly Asp Val Val Ser Ala Ile Arg Ala Gly Gly Asp 210 215 220 Glu Thr Lys Leu Leu Val Val Asp Arg Glu Thr Asp Glu Phe Phe Lys 225 230 235 240 Lys Cys Arg Val Ile Pro Ser Gln Glu His Leu Asn Gly Pro Leu Pro 245 250 255 Val Pro Phe Thr Asn Gly Glu Ile Gln Lys Glu Asn Ser Arg Glu Ala 260 265 270 Leu Ala Glu Ala Ala Leu Glu Ser Pro Arg Pro Ala Leu Val Arg Ser 275 280 285 Ala Ser Ser Asp Thr Ser Glu Glu Leu Asn Ser Gln Asp Ser Pro Pro 290 295 300 Lys Gln Asp Ser Thr Ala Pro Ser Ser Thr Ser Ser Ser Asp Pro Ile 305 310 315 320 Leu Asp Phe Asn Ile Ser Leu Ala Met Ala Lys Glu Arg Ala His Gln 325 330 335 Lys Arg Ser Ser Lys Arg Ala Pro Gln Met Asp Trp Ser Lys Lys Asn 340 345 350 Glu Leu Phe Ser Asn Leu 355 

That which is claimed is:
 1. An isolated nucleic acid molecule consisting of a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:2; (b) a nucleotide sequence consisting of SEQ ID NO:1; (c) a nucleotide sequence consisting of SEQ ID NO:3; and (d) a nucleotide sequence that is completely complementary to a nucleotide sequence of (a)-(c).
 2. A nucleic acid vector comprising the nucleic acid molecule of claim.
 3. A vector according to claim 2, wherein said vector is selected from the group consisting of a plasmid, a virus, and a bacteriophage.
 4. A vector according to claim 2, wherein said isolated nucleic acid molecule is inserted into said vector in proper orientation and correct reading fame such that a polypeptide comprising SEQ ID NO:2 may be expressed by a cell transformed with said vector.
 5. A vector according to claim 4, wherein said isolated nucleic acid molecule is operatively linked to a promoter sequence.
 6. A host cell containing the vector of claim
 2. 7. A process for producing a polypeptide comprising culturing the host cell of claim 6 under conditions sufficient for the production of said polypeptide, and recovering said polypeptide.
 8. An isolated polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO:1.
 9. An isolated polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO:3. 